g quadruplex dna and rna: their roles in regulation of dna replication … · 2020-05-26 · 1...
TRANSCRIPT
1
G-quadruplex DNA and RNA their roles in regulation of DNA
replication and other biological functions
Hisao Masai and Taku Tanaka
From Department of Genome Medicine Tokyo Metropolitan Institute of Medical
Science 2-1-6 Kamikitazawa Setagaya-ku Tokyo 156-8506 Japan
Correspondence to
Hisao Masai
Tel +81-3-5316-3220
Fax +81-3-5316-3145
E-mail masai-hsigakukenorjp
Key words G-quadruplex DNA replication initiation replication timing transcription
epigenome chromatin regulation
A review article for BBRC special issue G-quadruplex
2
Abstract
G-quadruplex is one of the best-studied non-B type DNA that is now known to be
prevalently present in the genomes of almost all the biological species Recent studies
reveal roles of G-quadruplex (G4) structures in various nucleic acids and chromosome
transactions In this short article we will first describe recent findings on the roles of G4
in regulation of DNA replication G4 is involved in regulation of spatio-temporal
regulation of DNA replication through interaction with a specific binding protein Rif1
This regulation is at least partially mediated by generation of specific chromatin
architecture through Rif1-G4 interactions We will also describe recent studies showing
the potential roles of G4 in initiation of DNA replication Next we will present
showcases of highly diversified roles of DNA G4 and RNA G4 in regulation of nucleic
acid and chromosome functions Finally we will discuss how the formation of cellular
G4 could be regulated
3
Contents
1 Introduction
2 G4 and DNA replication
2-1 Regulation of DNA replication
2-2 G-quadruplex as an architectural basis for chromatin organization that negatively
regulates replication initiation
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
2-2-2 Mechanisms of replication timing regulation by Rif1 protein
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
2-2-4 Biochemical characterization of Rif1 protein
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of replication
initiation
3 G4 and chromosome transactions
3-1 G4 and telomere
3-2 G4 and genome instability
3-3 Mechanism of G4 recognition and unwinding by G4 helicases
3-4 G4 and transcription
3-5 G4 and epigenome regulation
3-6 Relevance of G4 to diseases
3-7 Novel G4 binding proteins
2-8 G4 nucleic acids and viruses
3-9 G4 and phase separation
3-10 Other important biological roles of RNA G4 DNA G4 and their binding proteins
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
5 Conclusions
4
1 Introduction
G-quadruplex is one of the non-B type nucleic acid structures which is now known to
be widely present on the genomes of almost all the species It is composed of stacked
planar structures made of four guanines (called G-quartet) G4 is formed on
single-stranded DNA with a sequence context ldquoGge3NxGge3NxGge3NxGge3(x=1~7)rdquo This
algorithm predicted the presence of 375000 potential G4 on the human genome [1-4]
but search with ldquoG4-seqrdquo that utilizes the nature of G4 to block DNA polymerase led to
identification of 716310 putative G4 on the human genome [5] Analyses of RNA-G4
with a similar approach using reverse transcriptase led to identification of a number of
potential G4 on mRNA [6] G4 can adopt different topologies depending on how the
four strands run with each other and its three-dimensional structures also vary A
number of G4 proteins that selectively bind to G4 have been described [7] Among them
helicases that are capable of unfolding the G4 structure have been shown to play crucial
roles in maintenance of chromosome stability or promotion of translation or splicing of
mRNA [8] Various G4s probably interact with selective interactors to serve for specific
biological functions
In the first part we will discuss the roles of G4 in regulation of DNA
replication including those in chromatin-mediated regulation DNA replication is one of
the most fundamental processes for cell growth Recent genome-wide mapping of
initiation sites of DNA replication in higher eukaryotes including human indicates close
association of G4-forming sequences with mapped origins [9-11] Physical evidence
suggests that G4 structure is required for initiation in cells [12] In Escherichia coli an
alternative mode of chromosome DNA replication dependent on RNA-DNA hybrid is
known to exist [13] The potential role of G4-forming sequences and G4 structure in
this mode of DNA replication would be of interest since transcription of G4-forming
sequences facilitates formation of RNA-DNA hybrid in conjunction with G4 structure
composed most likely of non-template DNA strand and transcribed RNA ([14-18])
Recent studies on chromatin structures in nuclei indicate the existence of
ordered chromatin architecture topologically associated domain (TAD) determined by
the Chromatin Conformation Capture method that may be intimately related to
transcriptional pattern and changes during development or differentiation [19]
Accumulating evidence indicates that temporal and spatial regulation of DNA
replication is determined by replication timing domain which may be related to TAD
5
[2021] Rif1 an evolutionally conserved nuclear factor was identified as a key
regulator for regulation of DNA replication timing through altering the chromatin
architecture [22] Rif1 binds specifically G4 and may hold together the chromatin fiber
through its G4 binding and multimization abilities generating chromatin compartment
that may be inhibitory for initiation These results point to important roles of G4 in both
positive and negative regulation of DNA replication
G4 appearing in both DNA and RNA plays pleiotropic roles in various DNA
and RNA transactions These include telomere regulation epigenome regulation
transcription translation splicing viral maintenance and functions transpositions
repair and recombination (Figure 1) The G4s and their binding proteins regulate
important biological responses including maintenance of genome stability stress
responses immunogulobulin class switch recombination In the second part we will
summarize recent findings on the roles of G4 in various biological reactions Finally we
will discuss how dynamic formation and disappearance (unfolding) of G4 may be
regulated in cells
2 G4 and DNA replication
2-1 Regulation of DNA replication
DNA replication is one of the most fundamental processes of cell growth which takes
6~8 hrs during cell cycle One of the most important rules for regulation of DNA
replication is that it occurs once and only once during one cell cycle [23] This is
achieved by multiple layers of mechanisms that ensure the assembly of pre-RC
(pre-Replicative Complex) only during G1 phase but not after the onset of DNA
synthesis pre-RC is generated on chromatin by sequential assembly of ORC Cdc6 and
Cdt1-MCM Cell cycle-specific degradation of Cdt1 and stabilization of its inhibitor
Geminin as well as phosphorylation dependent-degradation and chromatin dissociation
of pre-RC factors after S phase onset is a part of the mechanisms for inhibition of
rereplication At the G1-S transition Cdc7-Dbf4 kinase phosphorylates components of
pre-RC most notably Mcm and triggers assembly of an active replicative helicase
complex CMG [see reviews in Masai H and M Foiani DNA Replication From Old
Principles to New Discoveries 2018 Springer Singapore]
During S phase tens of thousands of replication origins are activated to
ensure the replication of the entire genome within the allotted time For this replication
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[2] Huppert JL Bugaut A Kumari S Balasubramanian S G-quadruplexes the beginning and end
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[3] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs in
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[5] Chambers VS Marsico G Boutell JM Di Antonio M Smith GP Balasubramanian S
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[6] Kwok CK Marsico G Sahakyan AB Chambers VS BalasubramanianS rG4-seq reveals
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[7] Braacutezda V Haacuteroniacutekovaacute L Liao JC Fojta M DNA and RNA quadruplex-binding proteins Int J
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[8] Sauer M Paeschke K G-quadruplex unwinding helicases and their function in vivo Biochem
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[9] Cayrou C Coulombe P Vigneron A Stanojcic S Ganier O Peiffer I Rivals E Puy A
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[10] Besnard E Babled A Lapasset L Milhavet O Parrinello H Dantec C Marin JM Lemaitre JM
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[12] Valton AL Hassan-Zadeh V Lema I Boggetto N Alberti P Saintomeacute C Riou JF Prioleau MN
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28
[13] Kogoma T Stable DNA replication interplay between DNA replication homologous
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of G-quadruplexes Nucleic Acids Res 42 (2014) 7236-7346
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[16] Xiao S Zhang JY Zheng KW Hao YH Tan Z Bioinformatic analysis reveals an evolutional
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[17] Zheng KW Xiao S Liu JQ Zhang JY Hao YH Tan Z Co-transcriptional formation of
DNARNA hybrid G-quadruplex and potential function as constitutional cis element for
transcription control Nucleic Acids Res 41 (2013) 5533-5541
[18] Zhang C Liu HH Zheng KW Hao YH Tan Z DNA G-quadruplex formation in response to
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[20] Ryba T et al Evolutionarily conserved replication timing profiles predict long-range
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a010132
[22] Yamazaki S Hayano M Masai H Replication timing regulation of eukaryotic replicons Rif1
as a global regulator of replication timing Trends Genet 29 (2013) 449-460
[23] Masai H Matsumoto S You Z Yoshizawa-Sugata N Oda M Eukaryotic chromosome DNA
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[24] Ge XQ DA Jackson and JJ Blow Dormant origins licensed by excess Mcm2-7 are
required for human cells to survive replicative stress Genes Dev 21 (2007) 3331-3341
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Forkhead transcription factors establish origin timing and long-range clustering in S cerevisiae
Cell 148 (2012) 99-111
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[26] Sima J Chakraborty A Dileep V Michalski M Klein KN Holcomb NP Turner JL Paulsen
MT Rivera-Mulia JC Trevilla-Garcia C Bartlett DA Zhao PA Washburn BK Nora EP Kraft
K Mundlos S Bruneau BG Ljungman M Fraser P Ay F Gilbert DM Identifying cis Elements
for Spatiotemporal Control of Mammalian DNA Replication Cell 176 (2019) 816-830
[27] Hayano M Kanoh Y Matsumoto S Renard-Guillet C Shirahige K Masai H Rif1 is a global
regulator of timing of replication origin firing in fission yeast Genes Dev 26 (2012) 137-150
[28] Yamazaki S Ishii A Kanoh Y Oda M Nishito Y Masai H Rif1 regulates the replication
timing domains on the human genome EMBO J 31 (2012) 3667-3677
[29] Cornacchia D Dileep V Quivy JP Foti R Tili F Santarella-Mellwig R Antony C Almouzni G
Gilbert DM Buonomo SB Mouse Rif1 is a key regulator of the replication-timing programme
in mammalian cells EMBO J 31 (2012) 3678-3690
[30] Hardy CF Sussel L Shore D A RAP1-interacting protein involved in transcriptional silencing
and telomere length regulation Genes Dev 6 (1992) 801-814
[31] Kanoh J Ishikawa F spRap1 and spRif1 recruited to telomeres by Taz1 are essential for
telomere function in fission yeast Curr Biol 11 (2001) 1624-1630
[32] Masai H Miyake T Arai K hsk1+ a Schizosaccharomyces pombe gene related to
Saccharomyces cerevisiae CDC7 is required for chromosomal replication EMBO J 14 (1995)
3094-3104
[33] Matsumoto S Hayano M Kanoh Y Masai H Multiple pathways can bypass the essential role
of fission yeast Hsk1 kinase in DNA replication initiation J Cell Biol 195 (2011) 387-401
[34] Hayano M Kanoh Y Matsumoto S Masai H Mrc1 marks early-firing origins and coordinates
timing and efficiency of initiation in fission yeast Mol Cell Biol 31 (2011) 2380-2391
[35] Xu L Blackburn EH Human Rif1 protein binds aberrant telomeres and aligns along anaphase
midzone microtubules J Cell Biol 167 (2004) 819ndash830
[36] Silverman J Takai H Buonomo SB Eisenhaber F de Lange T Human Rif1 ortholog of a yeast
telomeric protein is regulated by ATM and 53BP1 and functions in the S-phase checkpoint
Genes Dev 18 (2004) 2108-2119
[37] Alver RC Chadha GS Gillespie PJ Blow JJ Reversal of DDK-Mediated MCM
Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability
Independently of ATRChk1 Cell Rep 18 (2017) 2508-2520
[38] Sreesankar E Senthilkumar R Bharathi V Mishra RK Mishra K Functional diversification of
yeast telomere associated protein Rif1 in higher eukaryotes 13 (2012) 255
30
[39] Daveacute A Cooley C Garg M Bianchi A Protein phosphatase 1 recruitment by Rif1 regulates
DNA replication origin firing by counteracting DDK activity Cell Rep 7 (2014) 53-61
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Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
2
Abstract
G-quadruplex is one of the best-studied non-B type DNA that is now known to be
prevalently present in the genomes of almost all the biological species Recent studies
reveal roles of G-quadruplex (G4) structures in various nucleic acids and chromosome
transactions In this short article we will first describe recent findings on the roles of G4
in regulation of DNA replication G4 is involved in regulation of spatio-temporal
regulation of DNA replication through interaction with a specific binding protein Rif1
This regulation is at least partially mediated by generation of specific chromatin
architecture through Rif1-G4 interactions We will also describe recent studies showing
the potential roles of G4 in initiation of DNA replication Next we will present
showcases of highly diversified roles of DNA G4 and RNA G4 in regulation of nucleic
acid and chromosome functions Finally we will discuss how the formation of cellular
G4 could be regulated
3
Contents
1 Introduction
2 G4 and DNA replication
2-1 Regulation of DNA replication
2-2 G-quadruplex as an architectural basis for chromatin organization that negatively
regulates replication initiation
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
2-2-2 Mechanisms of replication timing regulation by Rif1 protein
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
2-2-4 Biochemical characterization of Rif1 protein
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of replication
initiation
3 G4 and chromosome transactions
3-1 G4 and telomere
3-2 G4 and genome instability
3-3 Mechanism of G4 recognition and unwinding by G4 helicases
3-4 G4 and transcription
3-5 G4 and epigenome regulation
3-6 Relevance of G4 to diseases
3-7 Novel G4 binding proteins
2-8 G4 nucleic acids and viruses
3-9 G4 and phase separation
3-10 Other important biological roles of RNA G4 DNA G4 and their binding proteins
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
5 Conclusions
4
1 Introduction
G-quadruplex is one of the non-B type nucleic acid structures which is now known to
be widely present on the genomes of almost all the species It is composed of stacked
planar structures made of four guanines (called G-quartet) G4 is formed on
single-stranded DNA with a sequence context ldquoGge3NxGge3NxGge3NxGge3(x=1~7)rdquo This
algorithm predicted the presence of 375000 potential G4 on the human genome [1-4]
but search with ldquoG4-seqrdquo that utilizes the nature of G4 to block DNA polymerase led to
identification of 716310 putative G4 on the human genome [5] Analyses of RNA-G4
with a similar approach using reverse transcriptase led to identification of a number of
potential G4 on mRNA [6] G4 can adopt different topologies depending on how the
four strands run with each other and its three-dimensional structures also vary A
number of G4 proteins that selectively bind to G4 have been described [7] Among them
helicases that are capable of unfolding the G4 structure have been shown to play crucial
roles in maintenance of chromosome stability or promotion of translation or splicing of
mRNA [8] Various G4s probably interact with selective interactors to serve for specific
biological functions
In the first part we will discuss the roles of G4 in regulation of DNA
replication including those in chromatin-mediated regulation DNA replication is one of
the most fundamental processes for cell growth Recent genome-wide mapping of
initiation sites of DNA replication in higher eukaryotes including human indicates close
association of G4-forming sequences with mapped origins [9-11] Physical evidence
suggests that G4 structure is required for initiation in cells [12] In Escherichia coli an
alternative mode of chromosome DNA replication dependent on RNA-DNA hybrid is
known to exist [13] The potential role of G4-forming sequences and G4 structure in
this mode of DNA replication would be of interest since transcription of G4-forming
sequences facilitates formation of RNA-DNA hybrid in conjunction with G4 structure
composed most likely of non-template DNA strand and transcribed RNA ([14-18])
Recent studies on chromatin structures in nuclei indicate the existence of
ordered chromatin architecture topologically associated domain (TAD) determined by
the Chromatin Conformation Capture method that may be intimately related to
transcriptional pattern and changes during development or differentiation [19]
Accumulating evidence indicates that temporal and spatial regulation of DNA
replication is determined by replication timing domain which may be related to TAD
5
[2021] Rif1 an evolutionally conserved nuclear factor was identified as a key
regulator for regulation of DNA replication timing through altering the chromatin
architecture [22] Rif1 binds specifically G4 and may hold together the chromatin fiber
through its G4 binding and multimization abilities generating chromatin compartment
that may be inhibitory for initiation These results point to important roles of G4 in both
positive and negative regulation of DNA replication
G4 appearing in both DNA and RNA plays pleiotropic roles in various DNA
and RNA transactions These include telomere regulation epigenome regulation
transcription translation splicing viral maintenance and functions transpositions
repair and recombination (Figure 1) The G4s and their binding proteins regulate
important biological responses including maintenance of genome stability stress
responses immunogulobulin class switch recombination In the second part we will
summarize recent findings on the roles of G4 in various biological reactions Finally we
will discuss how dynamic formation and disappearance (unfolding) of G4 may be
regulated in cells
2 G4 and DNA replication
2-1 Regulation of DNA replication
DNA replication is one of the most fundamental processes of cell growth which takes
6~8 hrs during cell cycle One of the most important rules for regulation of DNA
replication is that it occurs once and only once during one cell cycle [23] This is
achieved by multiple layers of mechanisms that ensure the assembly of pre-RC
(pre-Replicative Complex) only during G1 phase but not after the onset of DNA
synthesis pre-RC is generated on chromatin by sequential assembly of ORC Cdc6 and
Cdt1-MCM Cell cycle-specific degradation of Cdt1 and stabilization of its inhibitor
Geminin as well as phosphorylation dependent-degradation and chromatin dissociation
of pre-RC factors after S phase onset is a part of the mechanisms for inhibition of
rereplication At the G1-S transition Cdc7-Dbf4 kinase phosphorylates components of
pre-RC most notably Mcm and triggers assembly of an active replicative helicase
complex CMG [see reviews in Masai H and M Foiani DNA Replication From Old
Principles to New Discoveries 2018 Springer Singapore]
During S phase tens of thousands of replication origins are activated to
ensure the replication of the entire genome within the allotted time For this replication
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
377x200 - 330kB - RiboCluster Profilertrade 広がるRNhttpruomblcojpbioproductepigeneticsRNAwo画像は著作権で保護されている場合があります
Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
3
Contents
1 Introduction
2 G4 and DNA replication
2-1 Regulation of DNA replication
2-2 G-quadruplex as an architectural basis for chromatin organization that negatively
regulates replication initiation
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
2-2-2 Mechanisms of replication timing regulation by Rif1 protein
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
2-2-4 Biochemical characterization of Rif1 protein
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of replication
initiation
3 G4 and chromosome transactions
3-1 G4 and telomere
3-2 G4 and genome instability
3-3 Mechanism of G4 recognition and unwinding by G4 helicases
3-4 G4 and transcription
3-5 G4 and epigenome regulation
3-6 Relevance of G4 to diseases
3-7 Novel G4 binding proteins
2-8 G4 nucleic acids and viruses
3-9 G4 and phase separation
3-10 Other important biological roles of RNA G4 DNA G4 and their binding proteins
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
5 Conclusions
4
1 Introduction
G-quadruplex is one of the non-B type nucleic acid structures which is now known to
be widely present on the genomes of almost all the species It is composed of stacked
planar structures made of four guanines (called G-quartet) G4 is formed on
single-stranded DNA with a sequence context ldquoGge3NxGge3NxGge3NxGge3(x=1~7)rdquo This
algorithm predicted the presence of 375000 potential G4 on the human genome [1-4]
but search with ldquoG4-seqrdquo that utilizes the nature of G4 to block DNA polymerase led to
identification of 716310 putative G4 on the human genome [5] Analyses of RNA-G4
with a similar approach using reverse transcriptase led to identification of a number of
potential G4 on mRNA [6] G4 can adopt different topologies depending on how the
four strands run with each other and its three-dimensional structures also vary A
number of G4 proteins that selectively bind to G4 have been described [7] Among them
helicases that are capable of unfolding the G4 structure have been shown to play crucial
roles in maintenance of chromosome stability or promotion of translation or splicing of
mRNA [8] Various G4s probably interact with selective interactors to serve for specific
biological functions
In the first part we will discuss the roles of G4 in regulation of DNA
replication including those in chromatin-mediated regulation DNA replication is one of
the most fundamental processes for cell growth Recent genome-wide mapping of
initiation sites of DNA replication in higher eukaryotes including human indicates close
association of G4-forming sequences with mapped origins [9-11] Physical evidence
suggests that G4 structure is required for initiation in cells [12] In Escherichia coli an
alternative mode of chromosome DNA replication dependent on RNA-DNA hybrid is
known to exist [13] The potential role of G4-forming sequences and G4 structure in
this mode of DNA replication would be of interest since transcription of G4-forming
sequences facilitates formation of RNA-DNA hybrid in conjunction with G4 structure
composed most likely of non-template DNA strand and transcribed RNA ([14-18])
Recent studies on chromatin structures in nuclei indicate the existence of
ordered chromatin architecture topologically associated domain (TAD) determined by
the Chromatin Conformation Capture method that may be intimately related to
transcriptional pattern and changes during development or differentiation [19]
Accumulating evidence indicates that temporal and spatial regulation of DNA
replication is determined by replication timing domain which may be related to TAD
5
[2021] Rif1 an evolutionally conserved nuclear factor was identified as a key
regulator for regulation of DNA replication timing through altering the chromatin
architecture [22] Rif1 binds specifically G4 and may hold together the chromatin fiber
through its G4 binding and multimization abilities generating chromatin compartment
that may be inhibitory for initiation These results point to important roles of G4 in both
positive and negative regulation of DNA replication
G4 appearing in both DNA and RNA plays pleiotropic roles in various DNA
and RNA transactions These include telomere regulation epigenome regulation
transcription translation splicing viral maintenance and functions transpositions
repair and recombination (Figure 1) The G4s and their binding proteins regulate
important biological responses including maintenance of genome stability stress
responses immunogulobulin class switch recombination In the second part we will
summarize recent findings on the roles of G4 in various biological reactions Finally we
will discuss how dynamic formation and disappearance (unfolding) of G4 may be
regulated in cells
2 G4 and DNA replication
2-1 Regulation of DNA replication
DNA replication is one of the most fundamental processes of cell growth which takes
6~8 hrs during cell cycle One of the most important rules for regulation of DNA
replication is that it occurs once and only once during one cell cycle [23] This is
achieved by multiple layers of mechanisms that ensure the assembly of pre-RC
(pre-Replicative Complex) only during G1 phase but not after the onset of DNA
synthesis pre-RC is generated on chromatin by sequential assembly of ORC Cdc6 and
Cdt1-MCM Cell cycle-specific degradation of Cdt1 and stabilization of its inhibitor
Geminin as well as phosphorylation dependent-degradation and chromatin dissociation
of pre-RC factors after S phase onset is a part of the mechanisms for inhibition of
rereplication At the G1-S transition Cdc7-Dbf4 kinase phosphorylates components of
pre-RC most notably Mcm and triggers assembly of an active replicative helicase
complex CMG [see reviews in Masai H and M Foiani DNA Replication From Old
Principles to New Discoveries 2018 Springer Singapore]
During S phase tens of thousands of replication origins are activated to
ensure the replication of the entire genome within the allotted time For this replication
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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G-quadruplexes and regulates the epigenetic status of several gene promoters J Biol Chem 294
(2019) 17709-17722
[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
protein 1α interacts with parallel RNA and DNA G-quadruplexes Nucleic Acids Res 48 (2020)
682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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initiatemolecular cascades of disease Nature 507 (2014) 195-200
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
Brenton JD Brown GW Shah SP Cescon D Mak TW Caldas C Stirling PC Hieter P
Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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Cockayne syndrome group A and B proteins converge on transcription-linked resolution of
non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
Halder S Ryan A Jackson SP Ramadan K Kuznetsov SG Biroccio A Sale JE Tarsounas M
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
Cell 61 (2016) 449-460
[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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molecular abnormality in ATRX-deficient malignant glioma Nat Commun 10 (2019) 943
[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
Davidovich C Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
Beauvineau C Guetta C Jamin C Teulade-Fichou MP Faringhraeus R Voisset C Blondel M
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
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36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
3 UTR of LINE-1 elements stimulate retrotransposition Nat Struct Mol Biol 24 (2017)
243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
4
1 Introduction
G-quadruplex is one of the non-B type nucleic acid structures which is now known to
be widely present on the genomes of almost all the species It is composed of stacked
planar structures made of four guanines (called G-quartet) G4 is formed on
single-stranded DNA with a sequence context ldquoGge3NxGge3NxGge3NxGge3(x=1~7)rdquo This
algorithm predicted the presence of 375000 potential G4 on the human genome [1-4]
but search with ldquoG4-seqrdquo that utilizes the nature of G4 to block DNA polymerase led to
identification of 716310 putative G4 on the human genome [5] Analyses of RNA-G4
with a similar approach using reverse transcriptase led to identification of a number of
potential G4 on mRNA [6] G4 can adopt different topologies depending on how the
four strands run with each other and its three-dimensional structures also vary A
number of G4 proteins that selectively bind to G4 have been described [7] Among them
helicases that are capable of unfolding the G4 structure have been shown to play crucial
roles in maintenance of chromosome stability or promotion of translation or splicing of
mRNA [8] Various G4s probably interact with selective interactors to serve for specific
biological functions
In the first part we will discuss the roles of G4 in regulation of DNA
replication including those in chromatin-mediated regulation DNA replication is one of
the most fundamental processes for cell growth Recent genome-wide mapping of
initiation sites of DNA replication in higher eukaryotes including human indicates close
association of G4-forming sequences with mapped origins [9-11] Physical evidence
suggests that G4 structure is required for initiation in cells [12] In Escherichia coli an
alternative mode of chromosome DNA replication dependent on RNA-DNA hybrid is
known to exist [13] The potential role of G4-forming sequences and G4 structure in
this mode of DNA replication would be of interest since transcription of G4-forming
sequences facilitates formation of RNA-DNA hybrid in conjunction with G4 structure
composed most likely of non-template DNA strand and transcribed RNA ([14-18])
Recent studies on chromatin structures in nuclei indicate the existence of
ordered chromatin architecture topologically associated domain (TAD) determined by
the Chromatin Conformation Capture method that may be intimately related to
transcriptional pattern and changes during development or differentiation [19]
Accumulating evidence indicates that temporal and spatial regulation of DNA
replication is determined by replication timing domain which may be related to TAD
5
[2021] Rif1 an evolutionally conserved nuclear factor was identified as a key
regulator for regulation of DNA replication timing through altering the chromatin
architecture [22] Rif1 binds specifically G4 and may hold together the chromatin fiber
through its G4 binding and multimization abilities generating chromatin compartment
that may be inhibitory for initiation These results point to important roles of G4 in both
positive and negative regulation of DNA replication
G4 appearing in both DNA and RNA plays pleiotropic roles in various DNA
and RNA transactions These include telomere regulation epigenome regulation
transcription translation splicing viral maintenance and functions transpositions
repair and recombination (Figure 1) The G4s and their binding proteins regulate
important biological responses including maintenance of genome stability stress
responses immunogulobulin class switch recombination In the second part we will
summarize recent findings on the roles of G4 in various biological reactions Finally we
will discuss how dynamic formation and disappearance (unfolding) of G4 may be
regulated in cells
2 G4 and DNA replication
2-1 Regulation of DNA replication
DNA replication is one of the most fundamental processes of cell growth which takes
6~8 hrs during cell cycle One of the most important rules for regulation of DNA
replication is that it occurs once and only once during one cell cycle [23] This is
achieved by multiple layers of mechanisms that ensure the assembly of pre-RC
(pre-Replicative Complex) only during G1 phase but not after the onset of DNA
synthesis pre-RC is generated on chromatin by sequential assembly of ORC Cdc6 and
Cdt1-MCM Cell cycle-specific degradation of Cdt1 and stabilization of its inhibitor
Geminin as well as phosphorylation dependent-degradation and chromatin dissociation
of pre-RC factors after S phase onset is a part of the mechanisms for inhibition of
rereplication At the G1-S transition Cdc7-Dbf4 kinase phosphorylates components of
pre-RC most notably Mcm and triggers assembly of an active replicative helicase
complex CMG [see reviews in Masai H and M Foiani DNA Replication From Old
Principles to New Discoveries 2018 Springer Singapore]
During S phase tens of thousands of replication origins are activated to
ensure the replication of the entire genome within the allotted time For this replication
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
5
[2021] Rif1 an evolutionally conserved nuclear factor was identified as a key
regulator for regulation of DNA replication timing through altering the chromatin
architecture [22] Rif1 binds specifically G4 and may hold together the chromatin fiber
through its G4 binding and multimization abilities generating chromatin compartment
that may be inhibitory for initiation These results point to important roles of G4 in both
positive and negative regulation of DNA replication
G4 appearing in both DNA and RNA plays pleiotropic roles in various DNA
and RNA transactions These include telomere regulation epigenome regulation
transcription translation splicing viral maintenance and functions transpositions
repair and recombination (Figure 1) The G4s and their binding proteins regulate
important biological responses including maintenance of genome stability stress
responses immunogulobulin class switch recombination In the second part we will
summarize recent findings on the roles of G4 in various biological reactions Finally we
will discuss how dynamic formation and disappearance (unfolding) of G4 may be
regulated in cells
2 G4 and DNA replication
2-1 Regulation of DNA replication
DNA replication is one of the most fundamental processes of cell growth which takes
6~8 hrs during cell cycle One of the most important rules for regulation of DNA
replication is that it occurs once and only once during one cell cycle [23] This is
achieved by multiple layers of mechanisms that ensure the assembly of pre-RC
(pre-Replicative Complex) only during G1 phase but not after the onset of DNA
synthesis pre-RC is generated on chromatin by sequential assembly of ORC Cdc6 and
Cdt1-MCM Cell cycle-specific degradation of Cdt1 and stabilization of its inhibitor
Geminin as well as phosphorylation dependent-degradation and chromatin dissociation
of pre-RC factors after S phase onset is a part of the mechanisms for inhibition of
rereplication At the G1-S transition Cdc7-Dbf4 kinase phosphorylates components of
pre-RC most notably Mcm and triggers assembly of an active replicative helicase
complex CMG [see reviews in Masai H and M Foiani DNA Replication From Old
Principles to New Discoveries 2018 Springer Singapore]
During S phase tens of thousands of replication origins are activated to
ensure the replication of the entire genome within the allotted time For this replication
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[2] Huppert JL Bugaut A Kumari S Balasubramanian S G-quadruplexes the beginning and end
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[3] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs in
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[5] Chambers VS Marsico G Boutell JM Di Antonio M Smith GP Balasubramanian S
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[7] Braacutezda V Haacuteroniacutekovaacute L Liao JC Fojta M DNA and RNA quadruplex-binding proteins Int J
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[9] Cayrou C Coulombe P Vigneron A Stanojcic S Ganier O Peiffer I Rivals E Puy A
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[10] Besnard E Babled A Lapasset L Milhavet O Parrinello H Dantec C Marin JM Lemaitre JM
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[12] Valton AL Hassan-Zadeh V Lema I Boggetto N Alberti P Saintomeacute C Riou JF Prioleau MN
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28
[13] Kogoma T Stable DNA replication interplay between DNA replication homologous
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of G-quadruplexes Nucleic Acids Res 42 (2014) 7236-7346
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[16] Xiao S Zhang JY Zheng KW Hao YH Tan Z Bioinformatic analysis reveals an evolutional
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[17] Zheng KW Xiao S Liu JQ Zhang JY Hao YH Tan Z Co-transcriptional formation of
DNARNA hybrid G-quadruplex and potential function as constitutional cis element for
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[18] Zhang C Liu HH Zheng KW Hao YH Tan Z DNA G-quadruplex formation in response to
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[22] Yamazaki S Hayano M Masai H Replication timing regulation of eukaryotic replicons Rif1
as a global regulator of replication timing Trends Genet 29 (2013) 449-460
[23] Masai H Matsumoto S You Z Yoshizawa-Sugata N Oda M Eukaryotic chromosome DNA
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[24] Ge XQ DA Jackson and JJ Blow Dormant origins licensed by excess Mcm2-7 are
required for human cells to survive replicative stress Genes Dev 21 (2007) 3331-3341
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Forkhead transcription factors establish origin timing and long-range clustering in S cerevisiae
Cell 148 (2012) 99-111
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[26] Sima J Chakraborty A Dileep V Michalski M Klein KN Holcomb NP Turner JL Paulsen
MT Rivera-Mulia JC Trevilla-Garcia C Bartlett DA Zhao PA Washburn BK Nora EP Kraft
K Mundlos S Bruneau BG Ljungman M Fraser P Ay F Gilbert DM Identifying cis Elements
for Spatiotemporal Control of Mammalian DNA Replication Cell 176 (2019) 816-830
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regulator of timing of replication origin firing in fission yeast Genes Dev 26 (2012) 137-150
[28] Yamazaki S Ishii A Kanoh Y Oda M Nishito Y Masai H Rif1 regulates the replication
timing domains on the human genome EMBO J 31 (2012) 3667-3677
[29] Cornacchia D Dileep V Quivy JP Foti R Tili F Santarella-Mellwig R Antony C Almouzni G
Gilbert DM Buonomo SB Mouse Rif1 is a key regulator of the replication-timing programme
in mammalian cells EMBO J 31 (2012) 3678-3690
[30] Hardy CF Sussel L Shore D A RAP1-interacting protein involved in transcriptional silencing
and telomere length regulation Genes Dev 6 (1992) 801-814
[31] Kanoh J Ishikawa F spRap1 and spRif1 recruited to telomeres by Taz1 are essential for
telomere function in fission yeast Curr Biol 11 (2001) 1624-1630
[32] Masai H Miyake T Arai K hsk1+ a Schizosaccharomyces pombe gene related to
Saccharomyces cerevisiae CDC7 is required for chromosomal replication EMBO J 14 (1995)
3094-3104
[33] Matsumoto S Hayano M Kanoh Y Masai H Multiple pathways can bypass the essential role
of fission yeast Hsk1 kinase in DNA replication initiation J Cell Biol 195 (2011) 387-401
[34] Hayano M Kanoh Y Matsumoto S Masai H Mrc1 marks early-firing origins and coordinates
timing and efficiency of initiation in fission yeast Mol Cell Biol 31 (2011) 2380-2391
[35] Xu L Blackburn EH Human Rif1 protein binds aberrant telomeres and aligns along anaphase
midzone microtubules J Cell Biol 167 (2004) 819ndash830
[36] Silverman J Takai H Buonomo SB Eisenhaber F de Lange T Human Rif1 ortholog of a yeast
telomeric protein is regulated by ATM and 53BP1 and functions in the S-phase checkpoint
Genes Dev 18 (2004) 2108-2119
[37] Alver RC Chadha GS Gillespie PJ Blow JJ Reversal of DDK-Mediated MCM
Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability
Independently of ATRChk1 Cell Rep 18 (2017) 2508-2520
[38] Sreesankar E Senthilkumar R Bharathi V Mishra RK Mishra K Functional diversification of
yeast telomere associated protein Rif1 in higher eukaryotes 13 (2012) 255
30
[39] Daveacute A Cooley C Garg M Bianchi A Protein phosphatase 1 recruitment by Rif1 regulates
DNA replication origin firing by counteracting DDK activity Cell Rep 7 (2014) 53-61
[40] Hiraga S Alvino GM Chang F Lian HY Sridhar A Kubota T Brewer BJ Weinreich M
Raghuraman MK Donaldson AD Rif1 controls DNA replication by directing Protein
Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex Genes Dev 28
(2014) 372-383
[41] Hiraga SI Ly T Garzoacuten J Hořejšiacute Z Ohkubo YN Endo A Obuse 5 Boulton SJ Lamond AI
Donaldson AD Human RIF1 and protein phosphatase 1 stimulate DNA replication origin
licensing but suppress origin activation EMBO Rep 18 (2017) 403-419
[42] Kanoh Y Matsumoto S Fukatsu R Kakusho N Kono N Renard-Guillet C Masuda K Iida K
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Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
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EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
6
is compartmentalized both spatially and temporally [23] In the early S phase
replication occurs mainly in the interior of the nuclei and at minute uniformly scattered
foci During mid-late S phase replication occurs at larger fewer foci located at the
nuclear periphery and around nucleoli In late S phase replication occurs at large
limited numbers of foci that colocalize with heterochromatin
The genome segments containing the origins that fire during the same timing
of S phase are often called ldquoreplication domainsrdquo and their lengths range from several
hundred kb to Mb [20] The origins to be fired may be selected in a stochastic manner
within the same replication domain Pre-RCs are generated on chromosomes in excess
over those required for replicating the entire genome and those that are not used for
firing could serve as ldquoback-upsrdquo in case of unscheduled fork arrest [24]
Many factors affect the replication timing Generally local chromatin
structures affect the timing open chromatin (nucleosome-free chromatin) is associated
with early replication and closed chromatin (heterochromatin) with late replication
Manipulation of chromatin structures (eg induction of histone acetylation) can locally
induce early firing of origins A transcription factor may induce clustering of replication
origins for coordinated early replication in yeast [25] Early replication control
element (ERCEs) was identified in mouse ES cells which facilitates early replication
and active transcription within a compartmentalized domain of the chromosome [26] On
the other hand a factor negatively regulating early firing was identified In a mutant
lacking Rif1 protein both in yeast and mammals genome-wide and large scale alteration
of replication timing was observed [2227-29] as described in detail below
2-2 G-quadruplex as an architectural basis for chromatin organization that
negatively regulates replication initiation (Figure 2)
2-2-1 Identification of Rif1 as a conserved regulator of replication timing
Rif1 originally identified as a telomere binding factor in yeast [3031] was rediscovered
in a screen for bypass suppressors of the fission yeast hsk1 null mutant [25] hsk1+
encodes a fission yeast kinase related to Cdc7 [32] Earlier studies suggested that
mutants bypassing the requirement of hsk1 for DNA replication encode factors that
affect the replication timing [3334] In accordance with this expectation rif1∆ cells
that could vigorously support the growth of hsk1∆ cells exhibited striking change of
origin firing pattern 30 of the late-firing origins fired early and similarly 30 of
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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G-quadruplexes and regulates the epigenetic status of several gene promoters J Biol Chem 294
(2019) 17709-17722
[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
protein 1α interacts with parallel RNA and DNA G-quadruplexes Nucleic Acids Res 48 (2020)
682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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initiatemolecular cascades of disease Nature 507 (2014) 195-200
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
Brenton JD Brown GW Shah SP Cescon D Mak TW Caldas C Stirling PC Hieter P
Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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Cockayne syndrome group A and B proteins converge on transcription-linked resolution of
non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
Halder S Ryan A Jackson SP Ramadan K Kuznetsov SG Biroccio A Sale JE Tarsounas M
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
Cell 61 (2016) 449-460
[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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molecular abnormality in ATRX-deficient malignant glioma Nat Commun 10 (2019) 943
[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
Davidovich C Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
Beauvineau C Guetta C Jamin C Teulade-Fichou MP Faringhraeus R Voisset C Blondel M
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
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36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
3 UTR of LINE-1 elements stimulate retrotransposition Nat Struct Mol Biol 24 (2017)
243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
7
early-firing origins became late-firing in rif1∆ cells The ChIP analyses indicated the
binding of Rif1 to selected segments on the fission yeast chromosome interestingly
Rif1 bound preferentially to the segments in the vicinity of the late-firing origins that
are affected by Rif1 mutation This suggested a possibility that Rif1 binding to
chromosome inhibits firing of nearby origins [27] Rif1 homologs in higher eukaryotes
were not associated with telomeres and its depletion did not affect the telomere
maintenance [3536] In mammalian cells Rif1 depletion caused hyperphosphorylation
of Mcm2 and Mcm4 targets of phosphorylation by Cdc7 kinase and hypereplication in
early S phase [2837]
2-2-2 Mechanisms of replication timing regulation by Rif1protein
Subsequent studies indicated two mechanisms for this regulation One is through its
ability to recruit a phosphatase PP1 [37-41] All the Rif1 orthologs except for those
from plants carry a conserved motif to which PP1 binds Mutations of this motif results
in loss of replication timing regulation and hyperphosphorylation of Mcms the targets
of Cdc7 kinase The recruited PP1 inhibits initiation by dephosphorylating Mcm and
others that are crucial for initiation Rif1rsquos ability to generate specific chromatin
structures contributes to a long-range effect of Rif1 binding on origin activity This is
exemplified by deregulation of origin firing over ~100 kb segment by a mutation of a
single Rif1BS (Rif1 binding site)
Depletion of Rif1 in mammalian cells resulted in specific disappearance of
mid-S type replication foci pattern and persistent display of early-S phase replication
foci pattern until very late S phase when it is replaced by the late heterochromatin
replication foci pattern indicating that Rif1 is specifically required for generating mid-S
replication domains [26] Rif1 is preferentially localized at nuclear periphery as well as
around nucleoli This localization is reminiscent to the replication foci observed during
mid-to-late S phase Rif1 is biochemically fractionated into detergent- and
nuclease-insoluble fractions (nuclear matrix) cofractionated with LaminB1 protein
Rif1 is also localized in a close proximity of Lamin B1 protein at the nuclear periphery
[26]
2-2-3 Chromatin binding of Rif1 recognition and binding of G4
Initial ChIP-chip analyses of Rif1 binding sites in fission yeast revealed binding sites
along the three chromosomes (much less on the chromosome 3 the most of which is
replicated early S phase) and the binding is maximum during G1 and gradually
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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[16] Xiao S Zhang JY Zheng KW Hao YH Tan Z Bioinformatic analysis reveals an evolutional
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[22] Yamazaki S Hayano M Masai H Replication timing regulation of eukaryotic replicons Rif1
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[23] Masai H Matsumoto S You Z Yoshizawa-Sugata N Oda M Eukaryotic chromosome DNA
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29
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Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
8
decreased as cells move from S to G2 More accurate analyses with ChIP-seq identified
155 binding sites in G1S phase [42] The survey of the sequences of Rif1-bound regions
led to identification of a conserved motif sequence GNNCNANG(TA)GGGGG
(designated Rif1CS) Further analyses indicated a set of 35 strong Rif1BS normally
containing two or more Rif1CS Rif1CS contains a G-tract and appears twice or more
with an average of 100 bp interval In 75 of the case Rif1CSs appear in a head-to-tail
orientation generating multiple G-tracts on one strand Analyses of these sequences on
an acrylamide gel indicated that they generate slow-migrating forms upon prior heat
denaturation and they disappeared after mutagenesis of the G-tracts suggesting the
formation of specific nucleic acid structures in a G-tract-dependent manner
Furthermore they were stabilized in the presence of chemical ligands that are known to
stabilize G4 (G4 ligands see Nagasawarsquos article in this special issue) These results
strongly suggested a possibility that Rif1BS can adopt G4 structures and Rif1 may
recognize the structured DNA This possibility was examined by the DNA binding
assay with purified fission yeast Rif1 protein Gel shift assays with various forms of G4
DNA indeed have shown that Rif1 binds to parallel-type G4 DNA with high affinity
(Kd=~03nM) [42-44]
ChIP-seq and 4C analyses of Rif1 in mouse ES cells revealed that it binds to
late-replicating domains and restricts the interactions between different replication
timing domains during G1 phase Rif1 generally makes extensive and rather weak
contacts with the chromosomes [45] We detected strong bindings at selected locations
on the chromosomes and these binding sites contain G-tracts that are capable of
forming G4 (Yoshizawa et al unpublished data) Purified human and mouse Rif1
proteins also specifically bind to G4 [46]
2-2-4 Biochemical characterization of Rif1 protein
Rif1 is a highly oligomeric protein and biochemical evidence indicates it can bind to
multiple DNAs simultaneously Both fission yeast and mammalian Rif1 proteins carry
dual G4 binding domains one in the C-terminal segment and the other within the
HEAT repeat Each segment can bind to G4 but with reduced affinity compared to the
full-length polypeptide [4346] The C-terminal segment also contain oligomerization
activity In budding yeast Rif1 a C-terminal segment can form a tetramer [47] Origin
suppression activity as measured by the ability to suppress a hsk1 mutant is
compromised in a rif1 mutant deficient in chromatin binding In fission yeast Rif1
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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[92] Bochman ML Paeschke K Zakian VA DNA secondary structures stability and function of
G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
Brenton JD Brown GW Shah SP Cescon D Mak TW Caldas C Stirling PC Hieter P
Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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Cockayne syndrome group A and B proteins converge on transcription-linked resolution of
non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
Halder S Ryan A Jackson SP Ramadan K Kuznetsov SG Biroccio A Sale JE Tarsounas M
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
Cell 61 (2016) 449-460
[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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molecular abnormality in ATRX-deficient malignant glioma Nat Commun 10 (2019) 943
[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
Davidovich C Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
Beauvineau C Guetta C Jamin C Teulade-Fichou MP Faringhraeus R Voisset C Blondel M
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
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36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
Sci Rep 7 (2017) 2341
[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
biology meets biophysics Biophys Rev (2020) doi 101007s12551-020-00680-x
[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
mRNAs with G4-structures in untranslated regions Nat Commun10 (2019) 2421
[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
9
C-terminal 90 aa is sufficient to generate an oligomer A model has been presented on
how Rif1 may generate chromatin architecture through formation of chromatin loops
[414244] Localization of Rif1 at the nuclear periphery suggests that it generates
chromatin compartments along the nuclear membrane Indeed budding yeast Rif1 was
reported to be palmitoylated by Pfa4 palmitoyl transferase and this lipid modification
facilitated DSB repair by localizing the DSB repair proximal to the inner nuclear
membrane [4849] Similarly mammalian Rif1 may be modified by lipid for its
localization at the inner nuclear membrane Identification of Rif1 mutants defective in a
specific function of Rif1 (oligomerization inner nuclear membrane association and G4
binding) and analyses of their functions will clarify the mechanisms of Rif1-mediated
chromatin reorganization and its role in defining replication timing domains
2-3 G-quadruplex on RNA-DNA hybrid as a potential novel regulator of
replication initiation (Figure 2)
Genome-wide analyses of replication origins in higher eukaryotes have been conducted
with a hope that the results would answer long-standing questions of ldquoWhere are
replication origins (oris) in the human genome and how are they selectedrdquo
Genome-wide analysis of replication origins by nascent strand (NS)
purification method in Drosophila and mouse cells indicate that most CpG islands
(CGI) contain oris [91050] DNA synthesis starts at the borders of CGI suggestive of
a dual initiation event Oris contain a unique nucleotide skew around NS peaks
characterized by GT and CA overrepresentation at the 5 and 3 of ori sites
respectively Generally regions rich in oris are early replicating whereas
ori-poor regions are late replicating [91050] The same group reported the presence of
Origin G-rich Repeated Elements (OGRE) potentially forming G4 at most origins
These coincide with nucleosome-depleted regions located upstream of the initiation
sites which are associated with a labile nucleosome containing H3K64ac [51] Similar
enrichment of G4 forming sequences near the origins was observed with origin mapping
by initiation site sequencing (ini-seq) in which newly synthesized DNA is directly
labelled with digoxigenin-dUTP in a cell-free system [11] This result circumvents the
criticism that the potential G4 enrichment is an artifact of NS analyses caused by the
secondary structure of G4
Furthermore mutations affecting the G4 formation decreased the origin
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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as a global regulator of replication timing Trends Genet 29 (2013) 449-460
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MT Rivera-Mulia JC Trevilla-Garcia C Bartlett DA Zhao PA Washburn BK Nora EP Kraft
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Gilbert DM Buonomo SB Mouse Rif1 is a key regulator of the replication-timing programme
in mammalian cells EMBO J 31 (2012) 3678-3690
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and telomere length regulation Genes Dev 6 (1992) 801-814
[31] Kanoh J Ishikawa F spRap1 and spRif1 recruited to telomeres by Taz1 are essential for
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[33] Matsumoto S Hayano M Kanoh Y Masai H Multiple pathways can bypass the essential role
of fission yeast Hsk1 kinase in DNA replication initiation J Cell Biol 195 (2011) 387-401
[34] Hayano M Kanoh Y Matsumoto S Masai H Mrc1 marks early-firing origins and coordinates
timing and efficiency of initiation in fission yeast Mol Cell Biol 31 (2011) 2380-2391
[35] Xu L Blackburn EH Human Rif1 protein binds aberrant telomeres and aligns along anaphase
midzone microtubules J Cell Biol 167 (2004) 819ndash830
[36] Silverman J Takai H Buonomo SB Eisenhaber F de Lange T Human Rif1 ortholog of a yeast
telomeric protein is regulated by ATM and 53BP1 and functions in the S-phase checkpoint
Genes Dev 18 (2004) 2108-2119
[37] Alver RC Chadha GS Gillespie PJ Blow JJ Reversal of DDK-Mediated MCM
Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability
Independently of ATRChk1 Cell Rep 18 (2017) 2508-2520
[38] Sreesankar E Senthilkumar R Bharathi V Mishra RK Mishra K Functional diversification of
yeast telomere associated protein Rif1 in higher eukaryotes 13 (2012) 255
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[39] Daveacute A Cooley C Garg M Bianchi A Protein phosphatase 1 recruitment by Rif1 regulates
DNA replication origin firing by counteracting DDK activity Cell Rep 7 (2014) 53-61
[40] Hiraga S Alvino GM Chang F Lian HY Sridhar A Kubota T Brewer BJ Weinreich M
Raghuraman MK Donaldson AD Rif1 controls DNA replication by directing Protein
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[41] Hiraga SI Ly T Garzoacuten J Hořejšiacute Z Ohkubo YN Endo A Obuse 5 Boulton SJ Lamond AI
Donaldson AD Human RIF1 and protein phosphatase 1 stimulate DNA replication origin
licensing but suppress origin activation EMBO Rep 18 (2017) 403-419
[42] Kanoh Y Matsumoto S Fukatsu R Kakusho N Kono N Renard-Guillet C Masuda K Iida K
Nagasawa K Shirahige K Masai H Rif1 binds to G quadruplexes and suppresses replication
over long distances Nat Struct Mol Biol 22 (2015) 889-897
[43] Kobayashi S Fukatsu R Kanoh Y Kakusho N Matsumoto S Chaen S Masai H Both a
Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex
Binding and Origin Regulation by the Rif1 Protein Mol Cell Biol 39 (2019) e00364-18
[44] Masai H Fukatsu R Kakusho N Kanoh Y Moriyama K Ma Y Iida K Nagasawa K Rif1
promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization
activities Sci Rep 9 (2019) 8618
[45] Foti R Gnan S Cornacchia D Dileep V Bulut-Karslioglu A Diehl S Buness A Klein FA
Huber W Johnstone E Loos R Bertone P Gilbert DM Manke T Jenuwein T Buonomo SC
Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program Mol Cell
61 (2016) 260-273
[46] Moriyama K Yoshizawa-Sugata N Masai H Oligomer formation and G-quadruplex binding by
purified murine Rif1 protein a key organizer of higher-order chromatin architecture J Biol
Chem 293 (2018) 3607-3624
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
10
efficiency measured by short nascent DNA analyses in DT40 as well as in mammalian
cells [12] In this report authors discovered that the orientation of G4 determines the
precise position of the replication start site The results also indicate that a single G4 is
not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory
element which may be binding sites of transcription factors [12] More recently
multiple functional assays using the transient replication of EBV-based episome as well
as the replication in the Xenopus egg extracts suggested the requirement of G4
formation for efficient origin activity The latter suggested the role of G4 in in the
activation of pre-RC but not in its formation [52] Although these results suggest a
direct role of G4 in origin firing the precise mechanism is totally unknown The
purified human ORC protein was shown to bind to G4 in vitro [53] However its
significance in origin recognition and firing is currently unknown
Bacterial replicons are replicated through specific recognition of an origin
(replicator) by an initiator protein which permits highly efficient initiation event This
is a very robust system and occurs with nearly 100 efficiency On the other hand in E
coli another replication system that does not depend on the major initiator-ori system is
known to exist This mode of replication known as stable DNA replication (SDR)
occurs constitutively in RNaseH deficient mutant cells (rnhA) [13] This replication
depends on transcription RecA and a replication factor PriA a conserved DEHX-type
helicase [54]
The SDR mode of replication does not depend on DnaA-oriC and presumably
is initiated at multiple locations on the genome Indeed the genetic search for specific
origins for SDR suggested the absence of specific segments required for SDR The
efficient occurrence of SDR in rnhA mutant suggests that RNA-DNA hybrids are
important for this mode of replication The search for cellular RNA-DNA hybrids by
using a specific probe (human RNaseH1 lacking the catalytic activity) led to
identification of ~100 sites under RNaseH-deficient condition Comparison of
RNA-DNA hybrid loci with the predicted loci for candidate G4 showed 66 of the
putative G4 overlaps with RNA-DNA hybrids (Sagi et al unpublished data) Indeed it
has been reported that formation of RNA-DNA hybrid is strongly coupled with the
presence of G-tracts on the template DNA (on the non-template strand) It was proposed
that both transcribed RNA and non-template DNA strand participate in forming ldquohybrid
G4rdquo [55] Replication of ColE1-type plasmids can occur both in RNaseH+ and RNaseH-
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[2] Huppert JL Bugaut A Kumari S Balasubramanian S G-quadruplexes the beginning and end
of UTRs Nucleic Acids Res 36 (2008) 6260-6268
[3] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs in
human DNA Nucleic Acids Res 33 (2005) 2901-2907
[4] Bedrat A Lacroix L amp Mergny J-L Re-evaluation of G-quadruplex propensity with
G4Hunter Nucleic Acid Res 44 (2016) 1746ndash1759
[5] Chambers VS Marsico G Boutell JM Di Antonio M Smith GP Balasubramanian S
High-throughput sequencing of DNA G-quadruplex structures in the human genome Nat
Biotechnol 33 (2015) 877-881
[6] Kwok CK Marsico G Sahakyan AB Chambers VS BalasubramanianS rG4-seq reveals
widespread formation of G-quadruplex structures in the human transcriptome Nat Methods 13
(2016) 841-844
[7] Braacutezda V Haacuteroniacutekovaacute L Liao JC Fojta M DNA and RNA quadruplex-binding proteins Int J
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formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
11
cells [56] In the latter condition RNA-DNA hybrid may persist in the absence of
RNaseH where DNA replication is initiated The formation of this RNA-DNA hybrid
may also depend on G4 since mutagenesis of G-tracts within the transcribed segment
results in loss of replication activity (Fukatsu et al unpublished)
Mechanisms of replication initiation on RNADNA hybrid-G4 are still not
clear Replication initiation on pBR322 with purified proteins indicated the persistent
RNA-DNA hybrid at origin provides a loading signal for primosome on the displaced
lagging strand template which may promote the synthesis of the lagging strand [57]
However it is not clear how exactly an RNA-DNA hybrid structure is generated how it
is recognized by PriA to generate a replication fork and how a potential G4 structure
contributes to this process An alternative mode of genome replication was reported also
in arhcae Haloferax volcanii in the absence of normal origins Interestingly this mode
of replication initiates at multiple genomic loci and depends on the recombinase [58]
similar to the SDR mode of DNA replication in E coli
Formation of G4 is expected to be dynamic influenced by many factors
within cells (see the last chapter) Thus we expect that biological events regulated by
G4 may undergo very dynamic alterations This sort of ldquodynamicsrdquo may suit the needs
of cells to be flexible adaptive and robust in response to various external perturbations
It could possibly provide molecular basis for the stochastic nature of origin selection as
well as stochastic activation inactivation of transcription in various cells
3 G4 and chromosome transactions (see Supplementary Table S1 for summary)
3-1 G4 and telomere
G4 is most enriched at telomeres and may be protecting the chromosome ends At the
same time replication fork blocks expected to be induced by G4 at the telomeres may
be the sources of genomic instability
Cancer cells mutated in ATRX are known to maintain telomere length by the
homologous recombination (HR)-associated alternative lengthening of telomeres (ALT)
pathway ATRX suppresses the ALT pathway and shortens telomeres Importantly
ATRX-mediated ALT suppression depends on the histone chaperone DAXX ATRX
suppresses replication fork stalling a known trigger for HR at telomeres A G4 stabilizer
partially reverses the effect of ATRX suggesting that stabilization of G4 promotes ALT
It was proposed that loss of ATRX causes telomere chromatinization which results in
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
Implications for the antiviral activity of a G-quadruplex ligand Antiviral Res 118 (2015)
123-131
36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
Sci Rep 7 (2017) 2341
[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
biology meets biophysics Biophys Rev (2020) doi 101007s12551-020-00680-x
[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
(2017) 243-247
[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
Res47 (2019) 11746-11754
[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
3 UTR of LINE-1 elements stimulate retrotransposition Nat Struct Mol Biol 24 (2017)
243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
mRNAs with G4-structures in untranslated regions Nat Commun10 (2019) 2421
[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
2296-2309
[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
(2019) 3667-3679
[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
K Tateishi-Karimata H Sugimoto N Miyoshi D An anionic phthalocyanine decreases NRAS
expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
37
[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
1681-1691
[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
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13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
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[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
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[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
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[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
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[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
12
the persistence of G4 structures leading to replication fork stalling collapse HR and
subsequent recombination-mediated telomere synthesis in ALT cancers (Figure 3) It is
known that G4s are generally present in the nucleosome free regions [59] It is an
interesting possibility that ATRX may serve as an anti-G4 factor through its ability to
modify chromatin structures [60]
Replication protein A (RPA) prevents G4 formation at lagging-strand
telomeres to maintain the telomere length In fission yeast Rpa1-D223Y mutation
induces telomere shortening This mutation impairs lagging-strand telomere replication
and leads to the accumulation of secondary structures and recruitment of the
homologous recombination factor Rad52 with concomitant reduction of Pot1 and Ccq1
association at telomeres Expression of the budding yeast Pif1 that can unwind G4
rescues all the telomeric defects of the D223Y mutant human RPA with the identical
mutation is deficient in G4 binding Thus RPA prevents the formation of G4 at
lagging-strand telomeres to promote shelterin association and maintain telomeres [61]
Similarly CST (CTC1-STN1-TEN1) an RPA-like complex that can bind and disrupts
G4 structure associates with G-rich single-strand DNA and helps resolve replication
problems both at telomeres and chromosome arms [62] (Figure 3)
The helicase RTEL1 plays a role in the removal of telomeric DNA secondary
structures which is essential for preventing telomere fragility and loss (Figure 3) In the
absence of RTEL1 T loops are inappropriately resolved by the SLX4 nuclease complex
resulting in loss of the telomere as a circle Depleting SLX4 or blocking DNA
replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(--)
cells but failed to suppress telomere fragility Conversely stabilization of telomeric
G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient
cells but had no impact on TC formation or telomere loss It was proposed that RTEL1
performs two distinct functions at telomeres it disassembles T loops and also
counteracts telomeric G4-DNA structures which together ensure the dynamics and
stability of the telomere RTEL1 also facilitates the replication in other heterochromatin
regions (see below) [63]
It is known that the common oxidative lesion
8-oxo-78-dihydro-2-deoxyguanine (8-oxoG) regulates telomere elongation by human
telomerase When 8-oxoG is present in the dNTP pool as 8-oxodGTP telomerase
utilization of the oxidized nucleotide during telomere extension is mutagenic and
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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Tvardovskiy A Kunzelmann S Herrero J Bartke T Gamblin SJ Wilson JR Jenner RG
G-tract RNA removes Polycomb repressive complex 2 from genes Nat Struct Mol Biol 26
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[87] Mao SQ Ghanbarian AT Spiegel J Martiacutenez Cuesta S Beraldi D Di Antonio M Marsico G
Haumlnsel-Hertsch R Tannahill D Balasubramanian S DNA G-quadruplex structures mold the
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[88] Mendez-Bermudez A Lototska L Bauwens S Giraud-Panis MJ Croce O Jamet K Irizar A
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
Dash D Chowdhury S Telomere repeat-binding factor 2 binds extensively to extra-telomeric
G-quadruplexes and regulates the epigenetic status of several gene promoters J Biol Chem 294
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
protein 1α interacts with parallel RNA and DNA G-quadruplexes Nucleic Acids Res 48 (2020)
682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
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lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
13
terminates further elongation In contrast a preexisting 8-oxoG within the telomeric
DNA sequence promotes telomerase activity by destabilizing the G4 DNA structure
Thus telomerase is inhibited or stimulated depending on how 8-oxoG arises in
telomeres inhibited by insertion of oxidized nucleotides and activated by direct reaction
with free radicals [64]
Although G4 at telomeres is inhibitory for telomere extension by telomerase
and needs to be removed for telomere elongation as described above telomerase
appears to be able to cope with this on its own The core telomerase enzyme complex is
sufficient for partial G4 resolution and extension Human telomerase can extend
purified conformationally homogenous human telomeric G4 which is in a parallel
intermolecular conformation in vitro Thus telomere G4 in a specific conformation may
have some functional significance [65]
Other telomere binding proteins potentially recognizing G4 includes HMGB1
a ubiquitous non-histone protein depletion of which induces telomere damages [66]
(Figure 3)
3-2 G4 and genome instability
G4 can impede the progression of replication fork Extensive analyses of the ability of
G4 structures using yeast assays indicated short loop length with pyrimidine bases and
high thermal stability as critical determinants for the genomic instability induced by
G4-forming minisatellites [67]
Specific helicases can unfold G4 structures and facilitate the replication fork
movement at these ldquohard-to-replicaterdquo segments Pif1 is one of the best-studied G4
resolvases A significant subset of the Pif1 binding sites as determined by genome-wide
chromatin immunoprecipitation overlaps with G4 motifs Replication slowed in the
vicinity of these motifs and they were prone to undergo breakage in Pif1-deficient cells
These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to
prevent replication fork stalling and DNA breakage [68] (Figure 4)
Binding of Pif1 to cellular G4 may be assisted by other proteins A
co-transcriptional activator Sub1 of Saccharomyces cerevisiae is a G4-binding factor
and contributes to genome maintenance at G4-forming sequences Genome instability
linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly
augmented in the absence of Sub1 This can be suppressed by its DNA binding domain
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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558 (2018) 465-469
[80] Singh SP Soranno A Sparks MA Galletto R3 Branched unwinding mechanism of the Pif1
family of DNA helicases Proc Natl Acad Sci U S A 116 (2019) 24533-24541
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[83] Wu G Xing Z Tran EJ Yang D DDX5 helicase resolves G-quadruplex and is involved in
MYC gene transcriptional activation Proc Natl Acad Sci U S A 116 (2019) 20453-20461
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[84] Calabrese DR Chen X Leon EC Gaikwad SM Phyo Z Hewitt WM Alden S Hilimire TA
He F Michalowski AM Simmons JK Saunders LB Zhang S Connors D Walters KJ Mock
BA Schneekloth JS Jr Chemical and structural studies provide a mechanistic basis for
recognition of the MYC G-quadruplex Nat Commun 9 (2018) 4229
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[86] Beltran M Tavares M Justin N Khandelwal G Ambrose J Foster BM Worlock KB
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[87] Mao SQ Ghanbarian AT Spiegel J Martiacutenez Cuesta S Beraldi D Di Antonio M Marsico G
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
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35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
14
or the human homolog PC4 Sub1 interacts specifically with co-transcriptionally formed
G4 DNA in vivo Furthermore Sub1 interacts with the G4-resolving helicase Pif1
suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA [69]
Mms1 an E3 ubiquitin ligase complex protein was identified as a novel G4-DNA
binding protein and promotes Pif1 helicase binding to these regions to maintain genome
stability [70]
RecQ helicases are known to target G4 and resolve its structure BLM
helicase belongs to this group of helicase and is mutated in Bloom syndrome a cancer
predisposition disorder Cells from Bloom syndrome patients exhibit striking genomic
instability characterized by excessive sister chromatid exchange events (SCEs) The
mapping of the genomic locations of SCEs in the absence of BLM indicates striking
enrichment at coding regions specifically at sites of G4 motifs in transcribed genes
(Figure 5) Thus BLM protects against genome instability by suppressing
recombination at sites of G4 structures particularly in transcribed regions of the
genome [71]
Using a zinc-finger nuclease translocation reporter assays poly(ADP)ribose
polymerase 3 (PARP3) was identified as a promoter of chromosomal rearrangements
across human cell types PARP3 regulates G4 DNA that inhibits repair by
nonhomologous end-joining and homologous recombination (Figure 5) Chemical
stabilization of G4 DNA in PARP3-- cells leads to widespread DNA double-strand
breaks and lethality Thus PARP3 suppresses G4 DNA and facilitates DNA repair by
multiple pathways [72]
Recent in vitro experiments indicate strong correlation between G4 and
R-loops (RNA-DNA hybrid) It was shown that transcription of G-rich DNA generates
G4 structures composed of both non-sense DNA and transcribed RNA (hybrid G4) on
RNA-DNA hybrid [1516] Specific G4 ligands stabilize G4s and simultaneously
increase R-loop levels in human cancer cells Genome-wide mapping of R loops
showed that the studied G4 ligands likely cause the spreading of R loops to adjacent
regions containing G4 preferentially at 3-end regions of expressed genes
Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4
ligands in BRCA2-proficient and BRCA2-silenced cancer cells Thus G4 ligands can
induce DNA damage by an R loop-dependent mechanism [73]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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family of DNA helicases Proc Natl Acad Sci U S A 116 (2019) 24533-24541
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[84] Calabrese DR Chen X Leon EC Gaikwad SM Phyo Z Hewitt WM Alden S Hilimire TA
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BA Schneekloth JS Jr Chemical and structural studies provide a mechanistic basis for
recognition of the MYC G-quadruplex Nat Commun 9 (2018) 4229
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
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35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
377x200 - 330kB - RiboCluster Profilertrade 広がるRNhttpruomblcojpbioproductepigeneticsRNAwo画像は著作権で保護されている場合があります
Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
15
3-3 Mechanisms of G4 recognition and unwinding by G4 helicases
A number of proteins that interact with G4 have been reported Some of them are
helicases that are capable of unfolding the G4 structures There has been progress in
understanding the structural basis of the G4 recognition and unwinding by these
proteins (see also the article by Nakanishi and Seimiya in this issue)
DHX36 a DEAH-box helicase unwinds G4 DNA in an ATP-dependent
manner but unfolds G4 RNA in an ATP-independent manner which is followed by
ATP-dependent refolding The ATP-dependent activity of DHX36 arises from the RNA
tail rather than the G4 [74]
The structure of a bacterial RecQ DNA helicase bound to unwound G4
indicates that a guanine base from the unwound G4 is sequestered within a
guanine-specific binding pocket Disruption of the pocket in RecQ blocks G4
unwinding but not G4 binding or duplex DNA unwinding indicating its essential role
in structure-specific G4 resolution [75]
The three representative G4 resolvases DHX36 Bloom helicase (BLM) and
Werner helicase (WRN) exhibit distinct G4 conformation specificity but use a common
mechanism of repetitive unfolding that leads to disrupting G4 structure multiple times
in succession The same resolving activity is sufficient to dislodge a stably bound G4
ligand including BRACO-19 NMM and Phen-DC3 [76]
Fragile X Mental Retardation Protein (FMRP) plays a central role in the
development of several human disorders including Fragile X Syndrome (FXS) and
autism FMRP specifically interacts with G4 on mRNAs through an
arginine-glycine-rich (RGG) motif It recognizes a G4 RNA by a β-turn in the RGG
motif of FMRP [77]
A 18-amino acid G4-binding domain of DHX36 binds to a parallel DNA G4
through the terminal guanine base tetrad (G-tetrad) and clamps the G4 using
three-anchor-point electrostatic interactions between three positively charged amino
acids and negatively charged phosphate groups This mode of binding is strikingly
similar to that of most ligands interacting with specific G4 [78]
More recently co-crystal structure of DHX36 bound to a DNA with a G4 and
a 3 single-stranded DNA segment was reported The N-terminal DHX36-specific motif
folds into a DNA-binding-induced α-helix that together with the OB-fold-like
subdomain selectively binds parallel G4 G4 binding alone induces rearrangements of
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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MYC gene transcriptional activation Proc Natl Acad Sci U S A 116 (2019) 20453-20461
34
[84] Calabrese DR Chen X Leon EC Gaikwad SM Phyo Z Hewitt WM Alden S Hilimire TA
He F Michalowski AM Simmons JK Saunders LB Zhang S Connors D Walters KJ Mock
BA Schneekloth JS Jr Chemical and structural studies provide a mechanistic basis for
recognition of the MYC G-quadruplex Nat Commun 9 (2018) 4229
[85] Law MJ Lower KM Voon HP Hughes JR Garrick D Viprakasit V Mitson M De Gobbi M
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[86] Beltran M Tavares M Justin N Khandelwal G Ambrose J Foster BM Worlock KB
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[87] Mao SQ Ghanbarian AT Spiegel J Martiacutenez Cuesta S Beraldi D Di Antonio M Marsico G
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
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682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
377x200 - 330kB - RiboCluster Profilertrade 広がるRNhttpruomblcojpbioproductepigeneticsRNAwo画像は著作権で保護されている場合があります
Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
16
the helicase core by pulling on the single-stranded DNA tail these rearrangements
drive G4 unfolding one residue at a time [79]
Pif1 unwinds double-stranded DNA by a branched mechanism with two
modes of activity In the dominant mode only short stretches of DNA can be
processively and repetitively opened with reclosure of the DNA occurring by
mechanisms other than strand-switching In the other less frequent mode longer
stretches of DNA are unwound via a path that is separate from the one leading to
repetitive unwinding [80]
Three well-known G4 ligands (an oxazole telomestatin derivative
pyridostatin and PhenDC3) which thermally stabilize G4 at different levels were
shown to inhibit BLM helicase activity at similar levels (2-3 fold) Thus G4 ligands can
inhibit a G4 resolvase activity although the precise mechanism for this inhibition is not
known [81]
3-4 G4 and transcription
Potential G4 forming sequences are known to be enriched in the promoter regions of
actively transcribed genes most notably a number of oncogenes implicating the crucial
roles of G4 in regulation of gene expression [182]
MYC one of the most critical oncogenes forms a DNA G4 in its proximal
promoter region (MycG4) that functions as a transcriptional silencer However MycG4
is highly stable in vitro and its regulatory role would require active unfolding DDX5 a
DEAD-box RNA helicase is extremely proficient at unfolding MycG4-DNA DDX5
efficiently resolves MycG4-DNA in the absence of a single-stranded overhang and ATP
hydrolysis is not directly coupled to G4-unfolding by DDX5 G-rich sequences are
bound by DDX5 in vivo and DDX5 is enriched at the MYC promoter and activates
MYC transcription by resolving DNA and RNA G4s On the other hand G4 ligands
inhibit DDX5-mediated MYC activation Thus DDX5 is a therapeutic candidate of
Myc suppression for cancer intervention [83]
A small molecule DC-34 was developed which downergulates Myc
expression by a G4-dependent mechanism Inhibition by DC-34 is significantly greater
for MYC than for other G4-driven genes The design of G4-interacting small molecules
that can selectively control gene expression will have therapeutic merit [84]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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Cockayne syndrome group A and B proteins converge on transcription-linked resolution of
non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
Halder S Ryan A Jackson SP Ramadan K Kuznetsov SG Biroccio A Sale JE Tarsounas M
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
Cell 61 (2016) 449-460
[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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molecular abnormality in ATRX-deficient malignant glioma Nat Commun 10 (2019) 943
[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
Davidovich C Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
Class Switch Recombination Mol Cell67 (2017) 361-373
[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
Beauvineau C Guetta C Jamin C Teulade-Fichou MP Faringhraeus R Voisset C Blondel M
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
Implications for the antiviral activity of a G-quadruplex ligand Antiviral Res 118 (2015)
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36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
Sci Rep 7 (2017) 2341
[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
biology meets biophysics Biophys Rev (2020) doi 101007s12551-020-00680-x
[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
3 UTR of LINE-1 elements stimulate retrotransposition Nat Struct Mol Biol 24 (2017)
243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
mRNAs with G4-structures in untranslated regions Nat Commun10 (2019) 2421
[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
K Tateishi-Karimata H Sugimoto N Miyoshi D An anionic phthalocyanine decreases NRAS
expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
17
3-5 G4 and epigenome regulation
ATRX (α-thalassemia mental retardation X-linked) an X-linked gene of the SWISNF
family is required for α-globin expression and mutations in ATRX cause syndromal
mental retardation ATRX binds to tandem repeat (TR) sequences in both telomeres and
euchromatin Many of the TRs are G rich and predicted to form non-B DNA structures
(including G4) in vivo Indeed ATRX binds G4 structures in vitro Genes associated
with these TRs can be dysregulated when ATRX is mutated and the change in
expression is determined by the size of the TR producing skewed allelic expression
[85]
Polycomb repressive complex 2 (PRC2) preferentially binds G tracts within
nascent precursor mRNA (pre-mRNA) especially within predicted G4 structures In
cells chromatin-associated G-tract RNA removes PRC2 from chromatin leading to
H3K27me3 depletion from genes Targeting G-tract RNA to the tumor suppressor gene
CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces
senescence These data indicate a novel role of G4 RNA in removal of an epigenome
regulator from the chromatin [86] (Figure 6)
The presence of G4 structure is tightly associated with CpG islands (CGI)
hypomethylation in the human genome These G4 sites are enriched for DNA
methyltransferase 1 (DNMT1) occupancy Indeed DNMT1 exhibits higher binding
affinity for G4s as compared to duplex hemi-methylated or single-stranded DNA G4
structure itself rather than sequence inhibits DNMT1 enzymatic activity It was
proposed that G4 formation sequesters DNMT1 thereby protecting certain CGIs from
methylation and inhibiting local methylation [87]
Heterochromatin (eg pericentromeres centromeres and telomeres) impedes
replication fork progression eventually leading in the event of replication stress to
chromosome fragility aging and cancer The shelterin subunit TRF2 ensures
progression of the replication fork through pericentromeric heterochromatin but not
centromeric chromatin TRF2 binds to pericentromeric Satellite III sequences during S
phase and recruits the G4-resolving helicase RTEL1 to facilitate fork progression [88]
Genome-wide ChIP-Seq analyses of TRF2-bound chromatin led to
identification of thousands of TRF2-binding sites within the extra-telomeric genome
These sites are enriched in potential G4-forming DNA sequences TRF2 binding altered
expression and the epigenetic state of several target promoters indicated by histone
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
18
modifications Thus TRF2 regulates expression of genes outside telomeres in a
G4-dependent fashion [89]
The unstructured hinge domain necessary for the targeting of HP1α to
constitutive heterochromatin recognizes parallel G4 formed by the Telomeric
Repeat-containing RNA (TERRA) transcribed from the telomere Through this
mechanism TERRA can enrich HP1α at telomeres to maintain heterochromatin [90]
3-6 Relevance of G4 to diseases
G4 and other non-B type DNA structures are formed on sequences with characteristic
nucleotide repeats Expansion of simple triplet repeats (TNR) underlies more than 30
severe degenerative diseases These and other ldquorepeatrdquo syndromes may be associated
with formation of aberrant G4 or other unusual structures on DNA and or transcribed
RNA [91] Cellular G4s profoundly affect the genomic stability through impeding
replication fork progression at telomeres and at other chromosome arms eventually
leading to DSB cell death aging genomic alterations and cancer formation [92] The
specific G4 ligands can be exploited to suppress G4-induced genome instability or
specific cancer cell killing
CX-5461 a G-quadruplex stabilizer exhibits specific toxicity against BRCA
deficient cancer cells and polyclonal patient-derived xenograft models including
tumors resistant to PARP inhibition CX-5461 blocks replication forks and induces
ssDNA gaps or breaks The BRCA and NHEJ pathways are required for the repair of G4
ligand-induced DNA damage and failure to do so leads to lethality These data strongly
support the idea that G4 targeting in HR and NHEJ deficient cancer cells can be an
efficient therapeutic choice [93]
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused
by mutations in the CSA or CSB genes Loss of CSA or CSB leads to polymerase
stalling at non-B DNA in a neuroblastoma cell line in particular at G4 structures and
recombinant CSB can melt G4 structures (Figure 4) Stabilization of G4 structures
activates the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1) and leads to
accelerated aging in Caenorhabditis elegans Thus transcription-coupled resolution of
secondary structures may be a mechanism to repress spurious activation of a DNA
damage response [94]
Replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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Gilbert DM Buonomo SB Mouse Rif1 is a key regulator of the replication-timing programme
in mammalian cells EMBO J 31 (2012) 3678-3690
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and telomere length regulation Genes Dev 6 (1992) 801-814
[31] Kanoh J Ishikawa F spRap1 and spRif1 recruited to telomeres by Taz1 are essential for
telomere function in fission yeast Curr Biol 11 (2001) 1624-1630
[32] Masai H Miyake T Arai K hsk1+ a Schizosaccharomyces pombe gene related to
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
19
significantly in cells lacking homologous recombination (HR) Treatment with the G4
ligand pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells
suggesting that G4 formation drives telomere instability PDS induces DSB
accumulation checkpoint activation and deregulated G2M progression and enhances
the replication defect intrinsic to HR deficiency reducing cell proliferation of
HR-defective cells PDS toxicity extends to HR-defective cells that have acquired
olaparib resistance through loss of 53BP1 or REV7 These results highlight the
therapeutic potential of G4-stabilizing ligand to selectively eliminate HR-compromised
cells and tumors including those resistant to PARP inhibition [95]
Inactivation of ATRX (α-thalassemia mental retardation X-linked) defines the
mutation in large subsets of malignant glioma Loss of ATRX induces replication stress
and DNA damage by way of G4 DNA ATRX deficiency in conjunction with chemical
G4 stabilization enhances DNA damage and cell death Other DNA-damaging therapies
including ionizing radiation synergize with ATRX-deficiency suggesting a potential
new strategies for drug development [96]
ATRX normally binds to G4 in CpG islands of the imprinted Xlr3b gene
inhibiting its expression by recruiting DNA methyltransferases Atrx mutation
upregulates Xlr3b expression in the mouse brain Xlr3b binds to dendritic mRNAs and
its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α
promoting synaptic dysfunction Treatment with 5-ALA which is converted into
G-quadruplex-binding metabolites in a body reduces RNA polymerase II recruitment
represses Xlr3b transcription and restores synaptic plasticity and cognitive proficiency
in ATRX model mice This suggests a potential therapeutic strategy to target G4 and
decrease cognitive impairment associated with ATRX syndrome [97]
3-7 Novel G4 binding proteins
Guanine-rich sequences are prevalent on both RNA and DNA and they have high
propensity to form G4 and frequently play important roles in regulation of various
chromatin events as stated above Recent reports continue to add new members to the
list of proteins that can bind to RNA DNA structures generated by these guanine-rich
sequences
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that
trimethylates H3K27 a mark of repressed chromatin The purified human PRC2 has a
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[80] Singh SP Soranno A Sparks MA Galletto R3 Branched unwinding mechanism of the Pif1
family of DNA helicases Proc Natl Acad Sci U S A 116 (2019) 24533-24541
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[84] Calabrese DR Chen X Leon EC Gaikwad SM Phyo Z Hewitt WM Alden S Hilimire TA
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BA Schneekloth JS Jr Chemical and structural studies provide a mechanistic basis for
recognition of the MYC G-quadruplex Nat Commun 9 (2018) 4229
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
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35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
20
high affinity for folded G4 structures but shows little binding to duplex RNAs G-tract
motifs are significantly enriched among PRC2-binding transcripts in vivo DNA
sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on
chromatin and H3K27me3-modified nucleosomes The results indicate the significance
of G4-like structures for epigenome regulation by PRC2 [98]
Activation-induced cytidine deaminase (AID) initiates both class switch
recombination (CSR) and somatic hypermutation (SHM) in antibody diversification In
vitro experiments indicated that G4-containing substrates mimicking the mammalian
immunoglobulin switch regions are particularly good AID substrates Crystal structures
of AID alone and that in complex with deoxycytidine monophosphate showed a
bifurcated substrate-binding surface that explains structured substrate recognition by
capturing two adjacent single-stranded overhangs simultaneously Moreover G4
substrates induce cooperative AID oligomerization Mutations disrupting bifurcated
substrate recognition or oligomerization compromise CSR in splenic B cells Thus G4
recognition of AID and its oligomerization plays a crucial role in CSR [99] For CSR
see also section 3-10
3-8 G4 nucleic acids and viruses
Viruses carry dynamic genomes which appear as duplex or single-stranded DNARNAs
as well as in other special forms of nucleic acids Therefore the chances of formation of
G4 on virus genomes and its transcripts are high The oncogenic Epstein-Barr virus
(EBV) evades the immune system by suppressing the essential EBNA1 protein to the
minimal level necessary for function while minimizing immune recognition EBNA1
contains the glycine-alanine repeats (GAr) which forms a G4 in its mRNA Nucleolin
(NCL) binds to the G4 RNA and inhibits the EBNA1 expression whereas its depletion
relieves the suppression Moreover the G-quadruplex ligand PhenDC3 prevents NCL
binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression
Thus the NCL-EBNA1 mRNA interaction is a promising therapeutic target to trigger an
immune response against EBV-carrying cancers [100]
The Herpes Simplex Virus-1 (HSV-1) genome contains multiple clusters of
repeated G-quadruplexes [101] Treatment of HSV-1 infected cells with a core extended
aphtalene diimide (c-exNDI) G4 ligand induced significant virus inhibition with no
cytotoxicity This was due to G4-mediated inhibition of viral DNA replication c-exNDI
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
21
preferentially recognizes HSV-1 G4s over cellular telomeric G4s [102]
Adeno-associated viruses (AAV) associate with the DNA binding protein
nucleophosmin (NPM1) Nucleophosmin was shown to interact with G4-forming
sequences in the AAV genome facilitating AAV production [103]
The long terminal repeat (LTR) of the proviral human immunodeficiency
virus (HIV)-1 genome is integral to virus transcription and host cell infection The
guanine-rich U3 region within the LTR promoter forms a parallel-stranded G4 which
stacks with neighboring G4 through its single-nucleotide bulges [104]
3-9 G4 and phase separation
A great deal of attention is being drawn to the membrane-less organelles observed in cells
which are now known to be formed by liquid-liquid phase separation (LLPS) caused by
proteins nucleic acids and their interactions [105] RNA associated with repeat
expansions can phase separate in vitro and in cells A hexanucleotide expansion
GGGGCC (G4C2) found in the C9ORF72 gene is the most common mutation
associated with amyotrophic lateral sclerosis and frontotemporal dementia
(C9-ALSFTD) ALSFTD GGGGCC repeat RNA (rG4C2) drives phase separations
The phase transition occurs at a similar critical repeat number as observed in the diseases
[105] Using rG4C2 as a model RNA phase separation was observed in vitro and in
cells in length- and structure-dependent manner It was also shown that G-quadruplex
structures are required for rG4C2-mediated phase separation Recent report indicates
that G4 RNA with more G-quartets and longer loops are more likely to form phase
separation These results suggest a possibility that G4 RNA can induce liquid phase
separation in cells although its physiological significance needs to wait for further
analyses [106-108]
3-10 Other important biological roles of RNA G4 DNA G4 and their binding
proteins
Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in
higher eukaryotes that constitute a large portion of the genomes Guanine-rich
sequences in the 3 untranslated regions (UTRs) of hominoid-specific LINE-1 elements
are coupled with retrotransposon speciation and contribute to retrotransposition through
the formation of G4 structures Stabilization of the G4 motif of a human-specific
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
377x200 - 330kB - RiboCluster Profilertrade 広がるRNhttpruomblcojpbioproductepigeneticsRNAwo画像は著作権で保護されている場合があります
Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
22
LINE-1 element by small-molecule ligands stimulates retrotransposition [109]
The targets of DHX36 one of the most potent G4 resolvases are
preferentially localized in stress granules DHX36 binds to G4-forming sequences on
mRNAs and reduces their abundance Knockout of DHX36 results in increase of stress
granule formation Thus DHX36 may facilitate the resolution of G4 RNA induced by
cellular stress [110]
RNA elements with G4-forming capacity promote exon inclusion Disruption
of G4-forming capacity abrogates exon inclusion G4 are enriched in heterogeneous
nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated
alternatively spliced exons in the human transcriptome Moreover hnRNPF regulates an
epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a
G4-dependent manner resulting in inhibition of EMT These data suggest a critical role
for RNA G4 in regulating alternative splicing [111] Small molecules were identified
that disrupt RNA G4 and inhibit G4-dependent alternative splicing [112]
An anionic phthalocyanine ZnAPC binds to a G4-forming oligonucleotide
derived from the 5-untranslated region of N-RAS mRNA resulting in selective
cleavage of the target RNAs G4 upon photo-irradiation Upon photo-irradiation
ZnAPC decreases N-RAS mRNA and NRAS expression and thus viability of cancer
cells [113]
The formation of R-loop structures during CSR can occur as part of a
post-transcriptional mechanism targeting AID to IgH S-regions This depends on the
RNA helicase DDX1 which binds to G4 structures present in intronic switch transcripts
and converts them into S-region R-loops This recruits the cytidine deaminase enzyme
AID to S-regions promoting CSR Chemical stabilization of G4 RNA or the expression
of a DDX1 ATPase-deficient mutant reduces R-loop levels over S-regions The DDX 1
mutant acts as a dominant-negative protein and reduce CSR efficiency Thus S-region
transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH
locus by the action of DDX1 [114]
The mitochondrial transcription factor A (TFAM) a high-mobility group
(HMG)-box protein plays a critical role in its expression and maintenance TFAM binds
to DNA G4 or RNA G4 TFAM multimerizes in a manner dependent on its G4 binding
Thus TFAM-G4 interaction is important for mitochondrial DNA replication and
transcription [115]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
[1] Huppert JL Balasubramanian S G-quadruplexes in promoters throughout the human genome
Nucleic Acids Res 35 (2007) 406-413
[2] Huppert JL Bugaut A Kumari S Balasubramanian S G-quadruplexes the beginning and end
of UTRs Nucleic Acids Res 36 (2008) 6260-6268
[3] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs in
human DNA Nucleic Acids Res 33 (2005) 2901-2907
[4] Bedrat A Lacroix L amp Mergny J-L Re-evaluation of G-quadruplex propensity with
G4Hunter Nucleic Acid Res 44 (2016) 1746ndash1759
[5] Chambers VS Marsico G Boutell JM Di Antonio M Smith GP Balasubramanian S
High-throughput sequencing of DNA G-quadruplex structures in the human genome Nat
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Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
23
Heme is an essential cofactor for many enzymes but free heme is toxic and its
levels are tightly regulated G4 binds heme avidly in vitro and presumably in cells as
well Addition PhenDC3 a G4 ligand displaces G4-bound heme increasing the
expression of heme-regulated genes It was proposed that G4 sequesters heme to protect
cells from the pathophysiological consequences of free heme [116]
ROS-mediated oxidation of DNA (yielding 8-oxo-78-dihydroguanine [OG]
in gene promoters) is a signaling agent for gene activation When OG is formed in
guanine-rich potential G4-forming sequences in promoter-coding strands base excision
repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) is initiated yielding an abasic
site (AP) The AP enables melting of the duplex to unmask the G4-forming sequences
adopting a G4 fold in which apurinicapyrimidinic endonuclease 1 (APE1) binds but
inefficiently cleaves the AP for activation of vascular endothelial growth factor (VEGF)
or endonuclease III-like protein 1 (NTHL1) genes Thus G4 plays a role for epigenetic
regulation of transcription [117]
In contrast to DNA G4 RNA G4 may be more readily formed on G-rich
sequences once it is transcribed However in some cases additional step may be
required for forming RNA G4 It was reported that RNA G4 can be generated from
cleaved tRNA fragments which augments the formation of stress granules by
suppressing translation [118]
4 Dynamic regulation of formation and disruption of cellular G-quadruplex
Initial bioinformatics analyses indicated the presence of 375000 potential G4 forming
sequences on the human genome [12119] On the other hand analyses with ldquoG4-seqrdquo
method that utilizes the polymerase stop reaction by the presence of G4 revealed
716310 G4 structures and 451646 of them were not predicted by computational
methods [5] In contrast genome wide search of cellular G4 by G4-specific antibody
BG4 revealed the presence of 1496 and 10560 G4 in non-cancer cells (NHEK normal
human epidermal keratinocytes) and in cancer cells (HaCaT spontaneously immortal-
ized counterparts of NHEK) respectively [59] The increase of G4 in immortalized cells
may reflect the active transcription and relaxed chromatin compared to the
non-immortalized cells These numbers are significantly smaller than that predicted by
sequences and G4-seq suggesting that most of the predicted G4 are not formed in the
cells However this could be due to failure to maintain the structure during the detection
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
References
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[86] Beltran M Tavares M Justin N Khandelwal G Ambrose J Foster BM Worlock KB
Tvardovskiy A Kunzelmann S Herrero J Bartke T Gamblin SJ Wilson JR Jenner RG
G-tract RNA removes Polycomb repressive complex 2 from genes Nat Struct Mol Biol 26
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[87] Mao SQ Ghanbarian AT Spiegel J Martiacutenez Cuesta S Beraldi D Di Antonio M Marsico G
Haumlnsel-Hertsch R Tannahill D Balasubramanian S DNA G-quadruplex structures mold the
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[88] Mendez-Bermudez A Lototska L Bauwens S Giraud-Panis MJ Croce O Jamet K Irizar A
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
Dash D Chowdhury S Telomere repeat-binding factor 2 binds extensively to extra-telomeric
G-quadruplexes and regulates the epigenetic status of several gene promoters J Biol Chem 294
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
protein 1α interacts with parallel RNA and DNA G-quadruplexes Nucleic Acids Res 48 (2020)
682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
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Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
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Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
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[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
24
process andor to the limitation of the BG4 which may recognize only a subset of G4
structures
Among the 35 strong Rif1BS which form G4 in vitro and are very likely to
form G4 in cells as well only two overlaps with 446 potential G4-forming sequences
predicted from the computational analyses [42120] This strongly suggests 1) it is
difficult to precisely predict the G4 functional in cells simply by analyzing the
sequences and 2) there are different classes of G4 that may play differential roles in
distinct chromosome reactions presumably through interacting with specific G4 binding
proteins The sequences of Rif1BS which span close to 100 bp in some cases are
characterized by the presence of multiple stretches of long G-tract (5 or 6 guanines) and
unusually long loop sequences (more that 20 nt) The detailed nuclease mapping
demonstrated the presence of long loops in the G4 structure formed on the duplex DNA
containing Rif1BS [121] The formation of the higher order structure depends on the
G-tracts on the G-rich strand and there is no indication for the formation of an
intercalated motif (i-motif) on the complementary C-rich strand The presence of multiple
G-tracts within Rif1BS permits generation of an alternative G4 structure when some of
the G-tracts are mutated [121] This suggests a potential of G-rich G4-forming sequences
to adopt different configurations of DNA structures within cells in response to changes of
chromatin state or microenvironment The formation of G4 structure on the G-rich
sequences with potentially long loop segments was suggested also from the genome-wide
ldquoG4-seqrdquo analyses on the human genome [5]
The formation of G4 depends on the presence of monovalent cation such as
K+ or Na+ in the center of the G-quartet that has an anionic space formed by O6 of the
guanine residues Li+ does not support the G4 formation and rather destabilizes the G4
[122123] G4 formation depends on Hoogsteen hydrogen bonding in stead of the
Watson-Crick-type hydrogen bonding that is required for B-type double helix structure
[124]
In cells G4 needs to be formed in competition with duplex DNA form G4
could be formed during the process that can transiently generate single-stranded DNA
(Figure 7) DNA replication is likely to provide such opportunity Indeed many reports
support the notion that G4 formed on the lagging strand template strand during the
course of DNA replication can cause replication fork barrier that arrests the fork
progression A group of helicases are known that can unwind the G4 structure
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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and telomere length regulation Genes Dev 6 (1992) 801-814
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[33] Matsumoto S Hayano M Kanoh Y Masai H Multiple pathways can bypass the essential role
of fission yeast Hsk1 kinase in DNA replication initiation J Cell Biol 195 (2011) 387-401
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midzone microtubules J Cell Biol 167 (2004) 819ndash830
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Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability
Independently of ATRChk1 Cell Rep 18 (2017) 2508-2520
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yeast telomere associated protein Rif1 in higher eukaryotes 13 (2012) 255
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DNA replication origin firing by counteracting DDK activity Cell Rep 7 (2014) 53-61
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Raghuraman MK Donaldson AD Rif1 controls DNA replication by directing Protein
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Donaldson AD Human RIF1 and protein phosphatase 1 stimulate DNA replication origin
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Nagasawa K Shirahige K Masai H Rif1 binds to G quadruplexes and suppresses replication
over long distances Nat Struct Mol Biol 22 (2015) 889-897
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Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex
Binding and Origin Regulation by the Rif1 Protein Mol Cell Biol 39 (2019) e00364-18
[44] Masai H Fukatsu R Kakusho N Kanoh Y Moriyama K Ma Y Iida K Nagasawa K Rif1
promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization
activities Sci Rep 9 (2019) 8618
[45] Foti R Gnan S Cornacchia D Dileep V Bulut-Karslioglu A Diehl S Buness A Klein FA
Huber W Johnstone E Loos R Bertone P Gilbert DM Manke T Jenuwein T Buonomo SC
Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program Mol Cell
61 (2016) 260-273
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purified murine Rif1 protein a key organizer of higher-order chromatin architecture J Biol
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Shore D Thomauml NH Rif1 and Rif2 shape telomere function and architecture through
multivalent Rap1 interactions Cell 153 (2013) 1340-1353
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
25
[6886125] (Figure 4) These G4 helicases can counteract the G4 fork barrier enabling
the replication to continue (see other sections) It was reported that depletion of a
helicase resulted in generation of double-stranded DNA breaks and sensitivity of growth
to G4 ligands
Transcription likewise provides an opportunity of forming single-strand
Transcription of duplex DNA containing G-clusters on the non-template strand
generates slow-migrating form on a polyacrylamide gel This structure is sensitive to
RNaseH suggesting the presence of RNA-DNA hybrid Mutations of the guanines on
the non-template strand or transcribed RNA lead to loss of the structure Replacement of
the guanine on the non-template DNA or on the transcribed RNA with deaza-guanine
leads to loss of this form 7th nitrogen atom is crucial for Hoogsteen hydrogen bonding
which is a basis of G-quartet structure All these data are consistent with the idea that
RNA-DNA hybrid generated by transcription of guanine-rich double-stranded DNA
carries G4 composed of both RNA and DNA [13-17 126-129]
As stated above the formation of G4 depends on the presence of various
coordinating metals Its stability may be affected by temperature pH molecular
crowding in the cellular microenvironment (Figure 7) Epigenomic state such as CpG
methylation affects the stability of G4 (130-132) The presence of G4 was reported to be
associated with CpG island hypomethylation in the human genome This appears to be
caused by the ability of G4 to inhibit DNA methyltransferase 1 enzymatic activity [87]
Thus G4 and CpG methylation affect with each other Other factors influencing the G4
formation stability include local topology (superhelicity) or chromatin architecture
[133] Stability of G4 in vitro varies greatly depending on the sequence or the solution
condition In cells where nucleic acids exist with many complex factors and under
varied microenvironmental conditions some of them may be stably formed whereas
others may be unstable undergoing more dynamic transition between formation and
disruption
5 Conclusions
G4 appears both in DNA and RNA and potentially in mixed RNADNA on RNA-DNA
hybrids Recent studies show a varieties of biological roles associated with nucleic acids
forming G4 or its related structures In DNA replication replication timing during S
phase is regulated by an evolutionally conserved Rif1 protein which specifically binds
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
26
to G4 Rif1 may regulate chromatin architecture by tethering chromatin fiber through
binding to multiple G4 G4 has been reported to be associated with replication origins in
higher eukaryotes and evidence for its potential functional importance in initiation of
bacterial genome replication has been presented Thus G4 may serve as both genomic
marks for initiation of DNA replication and platforms for generating chromatin
compartments that are inhibitory for initiation of DNA replication
Diverse roles of DNA G4 and RNA G4 have been reported for many genome
and epigenome regulations as well as in RNA metabolism Proteins that bind to G4
exert various effects on chromosome events Some can stabilize G4 and others can
disrupt it by its dissolvase activity Some recruits other proteins to regulate the process
Some change the chromatin structures
A low molecular weight chemicals that specifically binds to G4 can be
exploited to regulate these processes Various chemicals are generated which can even
discriminate different G4 at different locations or G4 with different topology (see
Nagasawarsquos article in this special issue) These highly selective G4 ligands will be very
useful reagents that could be developed for efficient anti-cancer drugs (see
Nakanishi-Seimiyarsquos article in this special issue)
Acknowledgements
I would like to thank Naoko Kakusho for help in the preparation of this manuscript
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (A)
[Grant Numbers 23247031 and 26251004] to HM We also would like to thank the
Uehara Memorial Foundation and the Takeda Science Foundation for the financial
support of this study We would like to thank all the members of the laboratory for
helpful discussion
27
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[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
27
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
2296-2309
[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
(2019) 3667-3679
[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
K Tateishi-Karimata H Sugimoto N Miyoshi D An anionic phthalocyanine decreases NRAS
expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
37
[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
1681-1691
[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
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[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
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[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
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[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
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[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
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[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
28
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3 UTR of LINE-1 elements stimulate retrotransposition Nat Struct Mol Biol 24 (2017)
243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
mRNAs with G4-structures in untranslated regions Nat Commun10 (2019) 2421
[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
2296-2309
[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
(2019) 3667-3679
[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
K Tateishi-Karimata H Sugimoto N Miyoshi D An anionic phthalocyanine decreases NRAS
expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
37
[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
1681-1691
[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
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[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
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[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
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[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
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[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
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[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
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[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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EVB HIV
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
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proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
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[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
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[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
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proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
31
[49] Fontana GA Hess D Reinert JK Mattarocci S Falquet B Klein D Shore D Thomauml NH Rass
U Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane
Nat Commun 10 (2019) 2535
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
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[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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[92] Bochman ML Paeschke K Zakian VA DNA secondary structures stability and function of
G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
Brenton JD Brown GW Shah SP Cescon D Mak TW Caldas C Stirling PC Hieter P
Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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Cockayne syndrome group A and B proteins converge on transcription-linked resolution of
non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
[95] Zimmer J Tacconi EMC Folio C Badie S Porru M Klare K Tumiati M Markkanen E
Halder S Ryan A Jackson SP Ramadan K Kuznetsov SG Biroccio A Sale JE Tarsounas M
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
Cell 61 (2016) 449-460
[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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molecular abnormality in ATRX-deficient malignant glioma Nat Commun 10 (2019) 943
[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
Davidovich C Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
Beauvineau C Guetta C Jamin C Teulade-Fichou MP Faringhraeus R Voisset C Blondel M
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
G-quadruplexes of EBNA1 mRNA Nat Commun 8 (2017) 16043
[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex
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36
[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
Sci Rep 7 (2017) 2341
[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
nucleophosmin in AAV replication Virology 510 (2017) 46-54
[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
biology meets biophysics Biophys Rev (2020) doi 101007s12551-020-00680-x
[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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243-247
[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
mRNAs with G4-structures in untranslated regions Nat Commun10 (2019) 2421
[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
promotes alternative splicing via the RNA-binding protein hnRNPF Genes Dev 31 (2017)
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
modulators of alternative splicing by targeting RNA G-quadruplexes Nucleic Acids Res 47
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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expression by breaking down its RNA G-quadruplex Nat Commun 9 (2018) 2271
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
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[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
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[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
33
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[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
34
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[89] Mukherjee AK Sharma S Bagri S Kutum R Kumar P Hussain A Singh P Saha D Kar A
Dash D Chowdhury S Telomere repeat-binding factor 2 binds extensively to extra-telomeric
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[90] Roach RJ Garaviacutes M Gonzaacutelez C Jameson GB Filichev VV Hale TK Heterochromatin
protein 1α interacts with parallel RNA and DNA G-quadruplexes Nucleic Acids Res 48 (2020)
682-693
[91] Haeusler AR Donnelly CJ Periz G Simko EA Shaw PG Kim MS Maragakis NJTroncoso
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G-quadruplex structures Nat Rev Genet 13 (2012) 770-780
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
K Soong J Lin SC Tsai AH Osako T Algara T Saunders DN Wong J Xian J Bally MB
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Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
lethality in BRCA12 deficient tumours Nat Commun 8 (2017) 14432
[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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non-B DNA Proc Natl Acad Sci U S A 113 (2016) 12502-12507
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Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds Mol
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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[97] Shioda N Yabuki Y Yamaguchi K Onozato M Li Y Kurosawa K Tanabe H Okamoto N Era
T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[100] Lista MJ Martins RP Billant O Contesse MA Findakly S Pochard P Daskalogianni C
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Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[102] Callegaro S Perrone R Scalabrin M Doria F Palugrave G Richter SN A core extended
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
Promotes Phase Transitions In Vitro and in Cells Cell Rep 21 (2017) 3573-3584
[108] Zhang Y Yang M Duncan S Yang X Abdelhamid MAS Huang L Zhang H Benfey PN
Waller ZAE Ding Y G-quadruplex structures trigger RNA phase separation Nucleic Acids
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
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[113] Kawauchi K Sugimoto W Yasui T Murata K Itoh K Takagi K Tsuruoka T Akamatsu
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[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
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Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
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[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
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[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
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[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
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[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
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[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
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[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
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JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
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[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
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specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
35
[93] Xu H Di Antonio M McKinney S Mathew V Ho B ONeil NJ Santos ND Silvester J Wei V
Garcia J Kabeer F Lai D Soriano P Banaacuteth J Chiu DS Yap D Le DD Ye FB Zhang A Thu
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Balasubramanian S Aparicio S CX-5461 is a DNA G-quadruplex stabilizer with selective
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[94] Scheibye-Knudsen M Tseng A Borch Jensen M Scheibye-Alsing K Fang EF Iyama T Bharti
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[96] Wang Y Yang J Wild AT Wu WH Shah R Danussi C Riggins GJ Kannan K Sulman EP
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T Sugiyama H Wada T Fukunaga K Nat Med 24 (2018) 802-813
[98] Wang X Goodrich KJ Gooding AR Naeem H Archer S Paucek RD Youmans DT Cech TR
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[99] Qiao Q Wang L Meng FL Hwang JK Alt FW Wu H AID Recognizes Structured DNA for
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[101] Artusi S Nadai M Perrone R Biasolo MA Palugrave G Flamand L Calistri A Richter SN
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[103] Satkunanathan S Thorpe R Zhao Y The function of DNA binding protein
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[104] De Nicola B Lech CJ Heddi B Regmi S Frasson I Perrone R Richter SN Phan AT
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[105] Yoshizawa T Nozawa RS Jia TZ Saio T Mori E Biological phase separation cell
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[106] Jain A Vale RD RNA phase transitions in repeat expansion disorders Nature 546
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[107] Fay MM Anderson PJ Ivanov P ALSFTD-Associated C9ORF72 Repeat RNA
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[109] Sahakyan AB Murat P Mayer C Balasubramanian S G-quadruplex structures within the
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[110] Sauer M Juranek SA Marks J De Magis A Kazemier HG Hilbig D Benhalevy D
Wang X Hafner M Paeschke K DHX36 prevents the accumulation of translationally inactive
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[111] Huang H Zhang J Harvey SE Hu X Cheng C RNA G-quadruplex secondary structure
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[112] Zhang J Harvey SE Cheng C A high-throughput screen identifies small molecule
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[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
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[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
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[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
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[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
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[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
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[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
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[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
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[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
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[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
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[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
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[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
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[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
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41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
36
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Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral
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Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
37
[114] Ribeiro de Almeida C Dhir S Dhir A Moghaddam AE Sattentau Q Meinhart A
Proudfoot NJ RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to
Promote IgH Class Switch Recombination Mol Cell 70 (2018) 650-662
[115] Lyonnais S Tarreacutes-Soleacute A Rubio-Cosials A Cuppari A Brito R Jaumot J Gargallo R
Vilaseca M Silva C Granzhan A Teulade-Fichou MP Eritja R Solagrave M The human
mitochondrial transcription factor A is a versatile G-quadruplex binding protein Sci Rep 7
(2017) 43992
[116] Gray LT Puig Lombardi E Verga D Nicolas A Teulade-Fichou MP Londontildeo-Vallejo A
Maizels N G-quadruplexes Sequester Free Heme in Living Cells Cell Chem Biol 26 (2019)
1681-1691
[117] Fleming AM Ding Y Burrows CJ Oxidative DNA damage is epigenetic by regulating
gene transcription via base excision repair Proc Natl Acad Sci U S A 114 (2017) 2604-2609
[118] Lyons SM Gudanis D Coyne SM Gdaniec Z Ivanov P Identification of functional
tetramolecular RNA G-quadruplexes derived from transfer RNAs Nat Commun 8 (2017)
1127
[119] Todd AK Johnston M Neidle S Highly prevalent putative quadruplex sequence motifs
in human DNA Nucleic Acids Res 33 (2005) 2901-2907
[120] Sabouri N Capra JA Zakian VA The essential Schizosaccharomyces pombe Pfh1 DNA
helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage BMC
Biol 12 (2014) 101
[121] Masai H Kakusho N Fukatsu R Ma Y Iida K Kanoh Y Nagasawa K Molecular
architecture of G-quadruplex structures generated on duplex Rif1-binding sequences J Biol
Chem 293 (2018) 17033-17049
[122] Gellert M Lipsett MN Davies DR Helix formation by guanylic acid Proc Natl Acad
Sci U S A 48 (1962) 2013-2018
[123] Sen D Gilbert W Guanine quartet structures Methods Enzymol 211 (1992) 191-199
[124] Sen D Gilbert W Formation of parallel four-stranded complexes by guanine-rich motifs
in DNA and its implications for meiosis Nature 334 (1988) 364-366
[125] WuY Shin-yaK and BroshRM Jr FANCJ helicase defective in Fanconia anemia and
breast cancer unwinds G-quadruplex DNA to defend genomic stability Mol Cell Biol 28
(2008) 4116ndash4128
[126] Xiao S Zhang JY Wu J Wu RY Xia Y Zheng KW Hao YH Zhou X Tan Z Formation
of DNARNA hybrid G-quadruplexes of two G-quartet layers in transcription expansion of the
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
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11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
38
prevalence and diversity of G-quadruplexes in genomes Angew Chem Int Ed Engl 53 (2014)
13110 ndash13114
[127] Liu JQ Xiao S Hao YH Tan Z Strand-Biased Formation of G-Quadruplexes in DNA
Duplexes Transcribed with T7 RNA Polymerase Angew Chem Int Ed Engl 54 (2015)
8992-8996
[128] Wu RY Zheng KW Zhang JY Hao YH Tan Z Formation of DNARNA hybrid
G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex
in mediating transcription termination Angew Chem Int Ed Engl 54 (2015) 2447-2451
[129] Zheng KW He YD Liu HH Li XM Hao YH Tan Z Superhelicity Constrains a
Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of
Transcription ACS Chem Biol 12 (2017) 2609-2618
[130] CC Hardin M Corregan BA Brown 2nd LN Frederick Cytosine-cytosine+ base
pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect
Biochemistry 32 (1993) 5870-5880
[131] B Zamiri M Mirceta K Bomsztyk RB Macgregor Jr CE Pearson Quadruplex
formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8(GGCCCC)8
repeat effect of CpG methylation Nucleic acids research 43 (2015) 10055-10064
[132] J Lin JQ Hou HD Xiang YY Yan YC Gu JH Tan D Li LQ Gu TM Ou
ZS Huang Stabilization of G-quadruplex DNA by C-5-methyl-cytosine in bcl-2 promoter
implications for epigenetic regulation Biochemical and biophysical research communications
433 (2013) 368-373
[133] SekiboDAT and FoxKR The effects of DNA supercoiling on G-quadruplex
formation Nucleic Acids Res 45 (2017) 12069ndash12079
[134] Mohaghegh P Karow JK Brosh RM Jr Bohr VA Hickson ID The Blooms and
Werners syndrome proteins are DNA structure-specific helicases Nucleic Acids Res 29 (2001)
2843-2849
[135] Johnson JE Cao K Ryvkin P Wang LS Johnson FB Altered gene expression in the
Werner and Bloom syndromes is associated with sequences having G-quadruplex forming
potential Nucleic Acids Res 38 (2010) 1114-1122
[136] Gray LT Vallur AC Eddy J Maizels N G quadruplexes are genomewide targets of
transcriptional helicases XPB and XPD Nat Chem Biol 10 (2014) 313-318
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
11 ページhttpswwwncbinlmnihgovcorelw20htmltileshop_pmctileshop_pmc_inlinehtmltitle=Click20on20image20to20zoomampp=PMC3ampid=5971202_gr7jpg
Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
39
[137] Siddiqui-Jain A Grand CL Bearss DJ Hurley LH Direct evidence for a G-quadruplex
in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc Natl Acad Sci U S A 99 (2002) 11593-11598
[138] Fernando H ReszkaAP Huppert J Ladame S Rankin S Venkitaraman AR Neidle S
Balasubramanian S A conserved quadruplex motif located in a transcription activation site of
the human c-kit oncogene Biochemistry 45 (2006) 7854-7860
[139] Cogoi S Paramasivam M Spolaore B Xodo LE Structural polymorphism within a
regulatory element of the human KRAS promoter formation of G4-DNA recognized by nuclear
proteins Nucleic Acids Res 36 (2008) 3765-3780
[140] Kang HJ Cui Y Yin H Scheid A Hendricks WPD Schmidt J SekulicA Kong D Trent
JM Gokhale V Mao H Hurley LH A Pharmacological Chaperone Molecule Induces Cancer
Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters J Am Chem Soc
138 (2016) 13673-13692
[141] Tauchi T Shin-ya K Sashida G Sumi M Okabe S Ohyashiki JH Ohyashiki K
Telomerase inhibition with a novel G-quadruplex-interactive agent telomestatin in vitro and in
vivo studies in acute leukemia Oncogene 25 (2006) 5719-5725
[142] Miyazaki T Pan Y Joshi K Purohit D Hu B Demir H Mazumder S Okabe S Yamori
T Viapiano M Shin-ya K Seimiya H Nakano I Telomestatin impairs glioma stem cell
survival and growth through the disruption of telomeric G-quadruplex and inhibition of the
proto-oncogene c-Myb Clin Cancer Res 18 (2012) 1268-1280
[143] Kumari S Bugaut A Huppert JL Balasubramanian S An RNA G-quadruplex in the 5
UTR of the NRAS proto-oncogene modulates translation Nat Chem Biol 3 (2007) 218-221
[144] Arora A Dutkiewicz M Scaria V Hariharan M Maiti S Kurreck J Inhibition of
translation in living eukaryotic cells by an RNA G-quadruplex motif RNA 14 (2008)
1290-1296
[145] Wolfe AL Singh K Zhong Y Drewe P Rajasekhar VK Sanghvi VR Mavrakis KJ
Jiang M Roderick JE Van der Meulen J Schatz JH Rodrigo CM Zhao C Rondou P de
Stanchina E Teruya-Feldstein J Kelliher MA Speleman F Porco JA Jr Pelletier J Raumltsch G
Wendel HG RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer
Nature 513 (2014) 65-70
[146] Weldon C Dacanay JG Gokhale V Boddupally PVL Behm-Ansmant I Burley GA
Branlant C Hurley LH Dominguez C Eperon IC Specific G-quadruplex ligands modulate the
alternative splicing of Bcl-X Nucleic Acids Res 46 (2018) 886-896
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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Click on image to magnify
transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
40
[147] BeaudoinJD Perreault JP Exploring mRNA 3-UTR G-quadruplexes evidence of roles
in both alternative polyadenylation and mRNA shortening Nucleic Acids Res 41 (2013)
5898-5911
[148] Subramanian M Rage F Tabet R Flatter E Mandel JL Moine H G-quadruplex RNA
structure as a signal for neurite mRNA targeting EMBO Rep 12 (2011) 697-704
[149] Guo JU Bartel DP RNA G-quadruplexes are globally unfolded in eukaryotic cells and
depleted in bacteria Science 353 (2016) pii aaf5371
[150] Cahoon LA Seifert HS An alternative DNA structure is necessary for pilin antigenic
variation in Neisseria gonorrhoeae Science 325 (2009) 764-767
[151] Cahoon LA Seifert HS Transcription of a cis-acting noncoding small RNA is required
for pilin antigenic variation in Neisseria gonorrhoeae PLoS Pathog 9 (2013) e1003074
[152] HuberMD LeeDC and MaizelsN G4 DNA unwinding by BLM and Sgs1p substrate
specificity and substrate-specific inhibition Nucleic Acids Res 30 (2002) 3954ndash3961
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
41
Legends to figures
Figure 1 Diverse biological functions of G-quadruplex (G4)
DNA G4 RNA G4 and G4 formed on RNA-DNA hybrid serve as important nucleic
acid signals for regulation of varieties of chromosome functions that involve DNA
RNA and complexes of RNA and DNA See text and also Supplementary Table S1 for
details
Figure 2 G4 regulates DNA replication both negatively and positively
Left Rif1 negatively regulates DNA replication by binding to G4 which may generate
chromatin architecture inhibitory for firing of origins Right In Ecoli
RNA-DNA hybrids containing G4 may serve as a signal for initiation of DNA
replication through the actions of proteins recognizing G4 Proteins such as PriA RecA
or an unknown G4 binding factor may play roles in initiation of DNA replication at the
G4 In higher eukaryotes G4-forming sequences are frequently associated with
replication origins
Figure 3 Regulation of telomere by G4 and its binding proteins
Formation of G4 in telomere inhibits replication of telomere causing genomic
instability Various proteins can counteract the formation of G4 in telomere RTEL1
helicase disrupts secondary structures at telomere and also disassembles T loops (not
shown in the figure) These two functions contribute to telomere stability Persistent
formation of G4 could result in ALT (alternative lengthening of telomeres) This is
especially apparent in cells lacking ATRX The ATRX induces loading of Histone H33
onto chromatin facilitating the chromatinization of telomere which inhibits ALT
Figure 4 Interference of transcription and replication by G4
Formation of G4 on a template strand can impede the movement of DNA polymerases
(upper red line) or RNA polymerases (lower blue line) Pif1 helicase disrupt the G4
structure on the DNA template strand and relieves the inhibition of DNA chain
elongation Sub1 or Mms1 facilitates the binding of Pif1 to G4 On the other hand CSB
is able to antagonize the block of transcription by G4
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
20200328 2344Click on image to zoom
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
42
Figure 5 Roles of PARP3 and Bloom in regulation of G4 on chromosomes
PARP3 destabilizes G4 and promotes repair of DSB through HR and NHEJ Bloom
helicase also disrupts G4 on transcribed coding segments preventing SCE which
occurs extensively in cells from Bloom Syndrome patients
Figure 6 Interaction of PRC2 with G4 in epigenome regulation
Polycomb repressive complex 2 (PRC2) induces 3K27me3 but is removed by G4 on
transcribed mRNA resulting in more active histone signature
Figure 7 Factors affecting the stability of G4 in cells
Various factors may affect the formation of cellular G4 Thus the formation and
disruption of G4 in cells may occur in a dynamic manner
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
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Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
RNA G4DNA G4transcription
13
Transcription
promotion or inhibition
Recombination
Bacteria Phase transition
Chromatin architecture
Chromatin loop
13
Epigenome
Coupling with replication
Retrotransposition
3ʼ-UTR LINE-1
20170421 1013m04_elementjpg 424times304 ピクセル
11 ページhttpwwwfaisorjpokadaokada-pastresearchkeywordsm04_elementjpg
EVB HIV
20170421 1043「翻訳 mRNA」の検索結果 - Yahoo検索(画像)
11 ページhttpssearchyahoocojpimagesearchp=翻訳+mRNAampei=UTF-8ampfr=top_ga1_samode3Ddetail26index3D1326st3D1
377x200 - 330kB - RiboCluster Profilertrade 広がるRNhttpruomblcojpbioproductepigeneticsRNAwo画像は著作権で保護されている場合があります
Translation
Inhibition
DNA replication
Replication initiation
ImmunoglobulinClass switch
Virus replicationmaintenance
ImmunoglobulinClass switch
Telomere
G4
Telomerase
DSB
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transposon
retroposon transposition
retropositionRNASwitch region
Masai amp Tanaka Figure 1
tandemrepeat(G4)
RNA G4DNA G4 Hybrid G4
Figure
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
染体構築
染体ループ
NSMB (2015)
Genome Res 2011 21(9)1438-49Nat Struct Mol Biol 2012 19(8)837-44
PLoS Genet 2014 10(5)e1004282EMBO J 2014 33(7) 732-746
Nucleic Acids Res 2016 44(21)10230-10247Nature Commun 2019 10(1)3274
Negative
G
GG
G
GG
GG
GGGG G
GG
GGG
G G GG G GG G G G G G
G
GG
G
GGG
G GG GG
GG
GGG GGGGGGGGGGGGG
Rif1
ssDNA
G-quadruplex
Rif1 - G-quadruplexള৬
minus 分裂酵母精製 Rif1
PP1
றત
PP1 Rif1ड़জ०ঐشਛ
Cdc7জথ৲
P
P
P
ളଲକਡ
P
କਡ
ളଲକਡ
Cdc
PPPPPPPPPPPPP
ളଲକਡ
c7৲
CdcজথPP
ളଲକਡ
P
A B
G4dephosphorylation
Association with nuclear membrane
後期複製起点
Early ori
Rif1 negatively regulates DNA replication by binding to G4
Early ori
late ori
late ori
oligomerization
Chromatin domains suppressive for DNA replication
phosphorylation
Positive
PriA
RecA
x
G4RNA-DNA hybrid could be a signal for initiation of DNA replication
Masai amp Tanaka Figure 2
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTEL1RPA
HMGB1
Telomere
CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG ATRX
DAXXHistonH33
ALTALT
Bloom
Masai amp Tanaka Figure 3
Chromatinization
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Pif1
GGG
GGG
GGG
GGG
CSACSB
Masai amp Tanaka Figure 4
Sub1Mms1
DNA replication
Transcription
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXXHistonH33
ALT
RPA
BloomPARP3
Chromosome arms
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
Repair by HR NHEJ
stabilization by G4 ligand Unwind
SCE(sister chromatid exchange)
transcribed coding segment
Masai amp Tanaka Figure 5
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
RTELRPA CST
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
GGG
ATRX
DAXX
HistonH33
ALT
RPaseI
Polycomb repressive complex 2 (PRC2)
H3K27me3
Polycomb target genes
Masai amp Tanaka Figure 6
Inactive active
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
TranscriptionReplication
DNA topologyEpigenome state
Chromosome localizationStabilizing proteins
Chromatin Environment
Metal ionpH
Molecular crowdingTemperature
Other factorsSequence
Destabilizing proteins
Masai amp Tanaka Figure 7
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
DNA G4 or RNA G4 Species (organella) Category of biological reactions Proteins involved (or others) G4 ligands involved Mechanisms Disease involved Literature
G4 DNA Human Telomere regulation ATRX (dependent on histone chaperoneDAXX)
Pyridostatin (PDS)ATRX suppresses the ALT pathway and shortens
telomeres ATRX loss causes telomerechromatinization with persistent G4
[60]
G4 DNA Fission yeast Telomere regulation RPA Pif1
RPA prevents the formation of G4 at lagging-strandtelomeres
Pif1 can unwind G4 and rescues all the telomericdefects of the RPA D223Y mutant
[61]
G4 DNA Human Telomere regulation RTEL1 SLX4RTEL1 removes telomeric DNA secondary
structures and ensures the dynamics and stability ofthe telomere
[63]
G4 DNA Human Telomere regulation 8-oxoG
8-oxoG present in the dNTP pool terminatestelomere elongation
a preexisting 8-oxoG within telomere DNAdestabilizes G4 and stimulate telomerase
[64]
DNAG4 (a parallel intermolecularconformation)
Human Telomere regulation core telomerase enzyme complex Sufficient for partial G4 resolution and extension [65]
G4 DNA Human Telomere regulation HMGB1 (a ubiquitous non-histone protein) Depletion induces telomere damages [60]
G4 DNA Human Telomere regulation CST (CTC1-STN1-TEN1) CST maintains genome integrity through resolution
of G4 structures[62]
DNA G4 (short loop length withpyrimidine bases)
Budding yeast Genome instability [67]
DNA G4 Fission yeast Genome instability Pif1Pif1 binding sites on chromatin overlap with G4sequences Replication slows and breaks in pif1∆
cells[68]
DNA G4(co-transcriptionally formed G4
DNA in Top1-deficient cells)Budding yeast Genome instability Sub1(human homolog PC4) Pif1
Contributes to genome maintenance at G4-formingsequences Interacts with the G4-resolving helicase
Pif1[69]
DNA G4 Human Genome instability PARP3 (poly(ADP)ribose polymerase 3 )
PyridostatinChemical stabilization of G4 DNA in
PARP3-- cells leads to widespread DNAdouble-strand breaks and lethality
PARP3 suppresses G4 DNA and facilitates DNArepair by multiple pathways
[72]
DNA G4 (on RNA-DNA hybrid) Human Genome instability
G4 ligands spread R loops to adjacent regionscontaining G4 preferentially at 3-end regions of
expressed genes G4 ligands can induce DNAdamage by an R loop-dependent mechanism (in
BRCA2+ and BRCA- cells)
[73]
DNA G4 Fission yeast Genome instability Mms1 (an E3 ubiquitin ligase complex protein) Binds G4 and promotes Pif1 helicase binding [70]
DNA G4 (in the transcribed regions) Human Genome instability BLM helicase Sister chromatid exchange events (SCEs) enrichedat G4 motifs in transcribed genes in BLM mutant
BLM suppressed recombination at sites of G4[71]
DNA G4 Human Helicase Bloom and Werner helicaseThe Blooms and Werners syndrome proteins arecapable of unwinding DNA with specific higher-order structures incluring G-quadruplex DNA
[134 152]
DNA G4 Human Unwinding mechanism DHX36Unwinds G4 DNA in an ATP-dependent manner
but unfolds G4 RNA in an ATP-independentmanner
[74]
DNA G4 Bacteria (Ecoli) Unwinding mechanism RecQ DNA helicase A guanine base from the unwound G4 is [75]
DNA G4 Human Unwinding mechanismDHX36 Bloom helicase (BLM) and Werner
helicase (WRN)
BRACO-19 NMM and Phen-DC3(stably bound G4 ligand dislodged by the
resolvases)
Distinct G4 conformation specificity but use acommon mechanism of repetitive unfolding thatleads to disrupting G4 structure multiple times in
succession
[76]
DNA G4 Human Unwinding mechanism DHX36 The N-terminal DHX36-specific motif folds into a [77]
DNA G4 Fisson yeast Unwinding mechanism Pif1Unwinds double-stranded DNA by a branched
mechanism with 2 modes of activity[80]
DNA G4 Human Unwinding mechanism BLM helicaseAn oxazole telomestatin derivative
pyridostatin and PhenDC3Inhibits BLM helicase activity at similar levels (2-3
fold)[81]
RNA G4 Human Mode of recognition FMRP (Fragile X Mental Retardation Protein)Specifically interacts with G4 on mRNAs through
an arginine-glycine-rich (RGG) motif Recognizes aG4 RNA by a β-turn in the RGG motif of FMRP
FXS(Fragile X Syndrome ) [77]
DNA G4 Human Mode of recognition DHX36
A 18-amino acid segement binds to a parallel DNAG4 through the terminal guanine base tetrad (G-
tetrad) and clamps the G4 using three-anchor-pointelectrostatic interactions between three positively
charged amino acids and negatively chargedphosphate groups
[78]
DNA G4 Human Transcription DDX5Inhibits DDX5-mediated MYC
activation
DDX5 unfolds MycG4-DNA DDX5 efficientlyresolves MycG4-DNA DDX5 is enriched at the
MYC promoter and activates MYC transcription byresolving G4 DNA and RNA
[83]
DNA G4 Human Transcription DC-34Downergulates Myc expression by a G4-dependent
mechanism Inhibition by DC-34 is significantlygreater for MYC than for other G4-driven genes
[84]
Supplementary Table 1 Summary of various functions of DNA G4 and RNA G4 as well their binding proteins and the effects of G4-specific ligands
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
DNA G4 Human Transcription
Gene expression is considerably altered in WS andBS cells as compared with normal and RTS cellsMoreover upregulated genes in WS and BS cellsare significantly enriched for PQS but not in RTS
cells
WS (Werner syndromes)BS (Bloom syndromes) and
RTS (RothmundndashThompson syndrome)
[135]
DNA G4 Human Transcription XPB and XPD
XPB and XPD overlap with G4 XPD is a robust G4DNA helicase and XPB binds G4 XPB and XPD
are enriched near the transcription start site ofhighly transcribed genes
XP [136]
DNA G4 Human Transcription C-myc TMPyP4
A specific G-quadruplex structure in the c-MYCpromoter functions as a transcriptional repressor
element TMPyP4 can stabilize the G4 and suppressthe transcription
[137]
DNA G4 Human Transcription C-kitThe c-kit promoter contains a G4 formng sequence
which appears to be conserved in mammalianspecies
[138]
DNA G4 Human Transcriptionheterogeneous nuclear ribonucleoprotein A1
K-ras
The human KRAS promoter contains two G4forming sequences at nuclease hypersensitive
element (NHE) which is bound byribonucleoprotein A1 in vitro
[139]
DNA G4 Human Epigenome regulation ATRXBinds to tandem repeat (TR) sequences in both
telomeres and euchromatin ATRX binds G4structures
[85]
DNA G4 Human epigenome regulation PRC2 (Polycomb repressive complex 2)
Binds G tracts within pre-mRNA PRC2 transfersfrom chromatin to pre-mRNA upon gene activationleading to H3K27me3 depletion from genesA novel
role of G4 RNA in removal of an epigenomeregulator from the chromatin
[86]
DNA G4 Human epigenome regulation DNMT1 (DNA methyltransferase 1 )
G4 is tightly associated with CpG islandshypomethylation DNMT1 bind G4s which inhibit
DNMT1 enzymatic activity G4 sequesters DNMT1and inhibits local methylation
[87]
DNA G4 Human Heterochromatin replication TRF2Binds to pericentromeric Satellite III sequences
during S phase and recruit G4-resolving helicaseRTEL1 to facilitate fork progression
[88]
DNA G4 Human Epigenome replication TRF2
TRF2 binding altered expression and the epigeneticstate of several target promoters indicated by
histone modifications TRF2 regulates expression ofgenes outside telomeres in a G4-dependent fashion
[89]
RNA G4 Human Heterochromatin regulation HP1αThe unstructured hinge domain of HP1α recognizesparallel G4 formed by TERRA TERRA can enrich
HP1α at telomeres to maintain heterochromatin[90]
RNA G4 Human Epigenome regulationPRC2(Polycomb repressive complex 2)
Histone methyltransferase that trimethylatesH3K27
PRC2 has a high affinity for G4 G-tract motifs areenriched at PRC2-binding RNA and DNA
(chromatin) and H3K27me3-modified nucleosomes[98]
DNA G4 Human Diseases-related (G4 therapy) CX-5461Blocks replication forks and induces ssDNA gaps or
breaks which will be repaired by the BRCA andNHEJ pathways
G4 targeting in HR andNHEJ deficient cancer cells
can be an efficienttherapeutic choice
[93]
DNA G4 Human C elegans Diseases-related CSACSB
pyridostatin and TMPYP4Stabilization of G4 activates the DNA
damage sensor PARP1 (poly-ADP ribpsepolymerase 1) and leads to accelerated
aging in Caenorhabditis elegans
Loss of CSA or CSB leads to polymerase stalling atG4 in a neuroblastoma cell line and CSB can melt
G4Cockayne syndrome [94]
DNA G4 Human Diseases-related
Pyridostatin (PDS)increases telomere fragility in BRCA2-
deficient cells (G4 formation drivestelomere instability)
PDS induces DSB accumulation checkpointactivation and deregulated G2M progression and
enhances the replication defect intrinsic to HRdeficiency
therapeutic potential of G4-stabilizing ligand to
selectively eliminate HR-compromised cells andtumors including those
resistant to PARP inhibition
[95]
DNA G4 Human Diseases-related ATRX CX-3543 (G4 stabilizer)
Loss of ATRX induces replication stress and DNAdamage by way of G4 DNA ATRX deficiency in
conjunction with chemical G4 stabilizationenhances DNA damage and cell death
malignant glyomaα-thalassemia mentalretardation X-linked
[96]
DNA G4 Human Diseases-related ATRX
5-ALA(Converted into G-quadruplex-binding
metabolites reduces RNA polymerase IIrecruitment represses Xlr3b transcription
and restores synaptic plasticity andcognitive proficiency in ATRX model
mice)
ATRX binds to G4 in CpG islands of the imprintedXlr3b gene inhibiting its expression by recruiting
DNA methyltransferases Atrx mutationupregulates Xlr3b expression in the mouse brain
ATRX syndrome [97]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
DNA G4 Human Telomere diseasesGTC365 (a small drug-likepharmacological chaperone
(pharmacoperone) molecule)
Promoter mutations in hTERT disrupt G4 andimpairs the silencing effect GTC365 induces the
correct folding of the mutant G4 reducing hTERTexpression causing cancer cell death and
senescence
[140]
DNA G4 mouse Telomerase diseases TelomestatinTelomesatin induced apoptose in leukemia cells in
a manner dependent on p38 MAP kinaseAcute Leukemia [141]
DNA G4 Human Telomerase diseases Telomestatin
Telomestatin inhibits growth of GSC (Glioma stemcells) through telomere disruption and c-Myb
inhibitionGBM (glioblastoma
multiforme)[142]
DNA G4 RNA G4 Human Neurodegenerative diseases nucleolin
GGGGCC repeats in C9orf72 forms DNA and RNAG4 and promotes RNAbullDNA hybrids (R-loops)Nucleolin preferentially binds to these strucures
and induce nucleolar stress
amyotrophic lateralsclerosis (ALS) and
frontotemporal dementia(FTD)
[91]
RNA G4 Human Epstein-Barr virus (EBV) Nucleolin
PhenDC3prevents NCL binding to EBNA1 mRNAand reverses GAr-mediated repression of
EBNA1 expression
EBV evades the immune system by suppressing theessential EBNA1 protein to the minimal level Theglycine-alanine repeats (GAr) in EBNA1 mRNA
form a G4 Nucleolin (NCL) binds to the G4 RNAand inhibits the EBNA1 expression whereas its
depletion relieves the suppression
[100]
DNA G4 Human Herpes Simplex Virus-1 (HSV-1)A core extended aphtalene diimide (c-
exNDI)Significant virus inhibition with no cytotoxicityG4-mediated inhibition of viral DNA replication
[102]
DNA G4 Human Adeno-associated viruses (AAV) Nucleophosmin (NPM)Interact with G4-forming sequences in
the AAV genome facilitating AAVproduction
[103]
G4 DNA Human human immunodeficiency virus (HIV)-1
The G-rich U3 region within the LTR promoterforms a parallel-stranded G4 which stacks with
neighboring G4 through its single-nucleotidebulges
[104]
RNA G4 Human Translation
Mature cytoplasmic tRNAs are cleaved duringstress response to produce tRNA fragments that
function to repress translation in vivo G4 RNA canbe generated from cleaved tRNA fragments which
augments the formation of stress granules bysuppressing translation
[118]
RNA G4 Human TranslationA highly conserved thermodynamically stable G4RNA in the 5-UTR of the human NRAS mRNA
suppresses its translationN-ras [143]
RNA G4 Human Translation Zic-1 zinc-finger protein G4 in the 5-UTR of mRNA inhibits translation in
vivo in eukaryotic cells[144]
RNA G4 Human Translation eIF4A RNA helicase
5 UTRs of select cancer genes (oncogenessuperenhancer-associated transcription factors andepigenetic regulators) conain G4 RNA that needs to
be unfolded by eIF4A RNA helicase
[145]
RNA G4 Human alternative splicinghnRNPF (heterogeneous nuclear
ribonucleoprotein F)
hnRNPF regulates an EMT-associated CD44isoform switch in a G4-dependent manner throughits recognitiono of G4 enriched in hnRNPF-binding
sites and near hnRNPF-regulated alternatively
[111]
RNA G4 Human Splicing ribonucleoprotein F (hnRNPF) emetine and cephaelineG4 on mRNA may be required for exon inclusion
through its interaction with hnRNPF Thus G4 RNAmay play an important role in alternative splicing
[112]
RNA G4 Human mRNA regulation HP1α An anionic phthalocyanine ZnAPC
Binds to a G4-forming oligonucleotide derived fromthe 5-untranslated region of NRAS mRNA
resulting in selective cleavages of the target RNAsG4 upon photo-irradiation
[113]
RNA G4 Human SplicingG4 RNA veryclose to splicing site inhibits the
binding of splicing factors[146]
RNA G4 Human mRNA metabolismG4 in 3-UTR affects alternative 3-endpolyadenylation and mRNA shortening
[147]
RNA G4 Human mRNA translocationG4 in 3-UTR affects mRNA translocation in
cortical neurites G4 may be a common neuritelocalization signal
[148]
DNA G4 Human Drosophila DNA replication67-90 of mammalian replication origins have
flanking G4 motifs[910115051]
DNA G4 Chicken DNA replication
G4 motifs were shown to be necessary for originfunction in two model origins in chicken DT40
cells and G4 stability correlates with originefficiency G4 orientation determines the precise
position of the replication start site
[12]
DNA G4 Human Xenopus DNA replicationG4 is required for initiation of DNA replicaition in
Xenopus egg extracts[1252]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]
DNA G4 Fission yeast DNA replication timing chromatin structure Rif1
Rif1 binds to chromain through recognition ofselective G4 sequences Rif1 forms chromatin loopsthrough binding to multiple G4 simultaneously This
may generate chromatin architecture that may beinhibitory for initiation of DNA replication
[4243]
DNA G4 RNA G4 Human DNA replication ORCHuman ORC binds preferentially to G4 DNA and
G4 RNA[53]
DNA G4 Neisseria gonorrhoeae Recombination RecA NMMG4-forming sequences play a role for formation ofnicks required for recombination for pilin antigenic
[150151]
DNA G4 HumanLong interspersed nuclear elements (LINEs) transposable
elements
PDS PhenDC3 12459 and PDC12Stabilization with the G4 ligands
stimulates retrotransposition
G-rich sequences in the 3 untranslated regions(UTRs) of LINE-1 elements are coupled withretrotransposon speciation and contribute to
retrotransposition through the formation of G4structures
[109]
DNA G4 Human Stress granule formation DHX36 helicase
DHX36 binds to G4-forming sequences on mRNAsand reduces their abundance Knockout of DHX36
results in increase of stress granule formationDHX36 may facilitate the resolution of G4 RNA
induced cellular stress
[110]
DNA G4 RNA G4 Human CSR (Class Switch Recpmbination) DDX1Pyridostatin reduces R-loop levels over
S-regions
During CSR DDX1 binds to G4 structures presentin intronic switch transcripts and converts them into
S-region R-loops This recruits AID to S-regionspromoting CSR S-region transcripts interconvertbetween G4 and R-loop structures to promote CSR
in the IgH locus by the action of DDX1
[111]
DNA G4 HumanClass switch recombination (CSR) and somatic
hypermutation (SHM)AID(Activation-induced cytidine deaminase)
G4-containing substrates are good AID substrates invitro G4 substrates induce cooperative AID
oligomerization Mutations disrupting bifurcatedsubstrate recognition or oligomerization
compromise CSR in splenic B cells G4 recognitionof AID and its oligomerization plays a crucial role
in CSR
[99]
DNA G4 RNA G4 Human Transcription ReplicationTFAM
(The mitochondrial transcription factor A)
The mitochondrial transcription factor A (TFAM) ahigh-mobility group (HMG)-box protein known to
play a critical role in its expression andmaintenance binds to G4 DNA or G4 RNA which
are prevalent in the mitochondrial genome
[115]
DNA G4 RNA G4 Human Regulation of HemePhenDC3 (displaces G4-bound heme
increasing the expression of heme-regulated genes)
Heme is an essential cofactor for many enzymesbut free heme is toxic and its levels are tightly
regulated G4 binds heme avidly in vitro PhenDC3a G-quadruplex ligand displaces G4-bound heme in
vitro and upregulating genes that support hemedegradation and iron homeostasis including hemeoxidase 1 the key enzyme in heme degradation
[116]
DNA G4 RNA G4 HumanEpigenetic regulation of transcription
Base excision repair8-oxo-78-dihydroguanine (OG)
APE1 (apurinicapyrimidinic endonuclease 1)
OG(8-oxo-78-dihydroguanine) formed in potentialG4-forming sequences triggers base excision repair(BER) by 8-oxoguanine DNA glycosylase (OGG1)yielding an abasic site (AP) AP melts duplex DNA
to facilitate G4 formation Apurinicapyrimidinicendonuclease 1 (APE1) binds but inefficiently
cleaves the AP for activation of vascular endothelialgrowth factor (VEGF) or endonuclease III-like
protein 1 (NTHL1) genes
[117]
DNA G4 Human Phase transition
ALSFTD GGGGCC repeat RNA (rG4C2) drives phaseseparations The phase transition occurs at a similar critical
repeat number as observed in the diseases G4 RNA with moreG-quartets and longer loops are more likely to form phase
separation
[105-107]
RNA G4 Mouse and EcoliEvidence is presented that show potential G4-RNAin mammalian cells is mostly unfolded On the otherhand G4-RNA can be formed in bacteria
[149]